Mutated fibroblast growth factor (fgf) 1 and methods of use

ABSTRACT

The present disclosure provides FGF1 mutant proteins, such as those having an N-terminal deletion, point mutation(s), or combinations thereof, which can reduce blood glucose in a mammal. Such mutant FGF1 proteins can be part of a chimeric protein that includes a β-Klotho-binding protein, an FGFR1c-binding protein, a β-Klotho-binding protein and a FGFR1c-binding protein, a C-terminal region from FGF19 or FGF21. In some examples, mutant FGF1 proteins have reduced mitogenic activity. Also provided are nucleic acid molecules that encode such proteins, and vectors and cells that include such nucleic acids. Methods of using the disclosed molecules to reduce blood glucose levels are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.61/893,766 filed Oct. 21, 2013, U.S. Provisional Application No.61/949,945 filed Mar. 7, 2014, U.S. Provisional Application No.61/975,530 filed Apr. 4, 2014, U.S. Provisional Application No.62/019,185 filed Jun. 30, 2014, and U.S. Provisional Application No.62/046,038 filed Sep. 4, 2014, all herein incorporated by reference.

ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant Nos.DK057978, DK090962, HL088093, HL105278 and ES010337 awarded by TheNational Institutes of Health, National Human Genome Research Institute.The government has certain rights in the invention.

FIELD

This application provides mutated FGF1 proteins, FGFR1c-binding proteinmultimers, nucleic acids encoding such proteins, and methods of theiruse, for example to treat a metabolic disease.

BACKGROUND

Type 2 diabetes and obesity are leading causes of mortality and areassociated with the Western lifestyle, which is characterized byexcessive nutritional intake and lack of exercise. A central player inthe pathophysiology of these diseases is the nuclear hormone receptor(NHR)PPARγ, a lipid sensor and master regulator of adipogenesis. PPARγis also the molecular target for the thiazolidinedione (TZD)-class ofinsulin sensitizers, which command a large share of the current oralanti-diabetic drug market. However, there are numerous side effectsassociated with the use of TZDs such as weight gain, liver toxicity,upper respiratory tract infection, headache, back pain, hyperglycemia,fatigue, sinusitis, diarrhea, hypoglycemia, mild to moderate edema, andanemia. Thus, the identification of new insulin sensitizers is needed.

SUMMARY

It is shown herein that mutants of fibroblast growth factor (FGF) 1having reduced or eliminated mitogenic activity can be used to reduceblood glucose in a mammal. Based on these observations, methods forreducing blood glucose in a mammal, for example to treat a metabolicdisease, are disclosed. Such FGF1 mutants can have an N-terminaltruncation, point mutations, or combinations thereof, for example toreduce the mitogenic activity of the native FGF1 protein. Such FGF1mutants can be used alone or in combination with other agents, such asother glucose reducing agents, such as thiazolidinedione.

In some examples, the FGF1 mutant is part of a chimeric protein, such asone that includes at least 10, at least 20, at least 30, at least 40, atleast 42, at least 43, at least 44, at least 45, at least 46, at least47, at least 48, at least 49, or at least 50 contiguous amino acids froma C-terminal end of FGF19 or FGF21.

In some examples, the FGF1 mutant is part of a chimeric protein, such asone that includes at least 10, at least 20, at least 30, at least 35, atleast 40, at least 50, at least 60, at least 70, at least 80, at least90, at least 100, at least 120, at least 150, at least 180, or at least200 amino acids (such as 20-500, 20 to 250, 30 to 200, 35 to 180, 37 to90, or 37 to 180 amino acids) of a protein that selectively binds tobeta-Klotho (β-Klotho), such as SEQ ID NO: 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145 or 146.

In some examples, the FGF1 mutant is part of a chimeric protein, such asone that includes at least 10, at least 20, at least 30, at least 35, atleast 40, at least 50, at least 60, at least 70, at least 80, at least90, at least 100, at least 120, at least 150, at least 180, or at least200 amino acids (such as 20-500, 20 to 250, 30 to 200, 35 to 180, 37 to90, or 37 to 180 amino acids) of a protein that selectively binds toFGFR1c, such as SEQ ID NO: 147, 148, 149, 150, 151, 152, 153, 154, 155,156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, or multimersthereof (e.g., dimers, timers), such as SEQ ID NO: 190.

In some examples, the FGF1 mutant is part of a chimeric protein, such asone that includes at least 10, at least 20, at least 30, at least 35, atleast 40, at least 50, at least 60, at least 70, at least 80, at least90, at least 100, at least 120, at least 150, at least 180, or at least200 amino acids (such as 20-500, 20 to 250, 30 to 200, 35 to 180, 37 to90, or 37 to 180 amino acids) of a protein that selectively binds toβ-Klotho, and that includes at least 10, at least 20, at least 30, atleast 35, at least 40, at least 50, at least 60, at least 70, at least80, at least 90, at least 100, at least 120, at least 150, at least 180,or at least 200 amino acids (such as 20-500, 20 to 250, 30 to 200, 35 to180, 37 to 90, or 37 to 180 amino acids) of a protein that selectivelybinds to FGFR1c, such as SEQ ID NO: 168, 169, 170 or 171.

In some examples, chimeric proteins include a linker between the FGF1mutant and the FGF19, FGF21, FGFR1c-binding, or β-Klotho-bindingsequence. In some examples, use of the disclosed methods result in oneor more of: reduction in triglycerides, decrease in insulin resistance,reduction of hyperinsulinemia, increase in glucose tolerance, orreduction of hyperglycemia in a mammal.

Provided herein are mutated FGF1 proteins, which can include deletion ofan N-terminal portion of FGF1, point mutations (such as amino acidsubstitutions, deletions, additions, or combinations thereof), orcombinations of N-terminal deletions and point mutations, and methods oftheir use to lower glucose, for example to treat a metabolic disease. Insome examples, such mutations reduce the mitogenicity of mature FGF1(e.g., SEQ ID NO: 5), such as a reduction of at least 20%, at least 50%,at least 75% or at least 90%. In some examples, the mutant FGF1 proteinis a truncated version of the mature protein (e.g., SEQ ID NO: 5), whichcan include for example deletion of at least 5, at least 6, at least 10,at least 11, at least 12, at least 13, or at least 20 consecutiveN-terminal amino acids. In some examples, one or more of the deletedN-terminal amino acids are replaced with corresponding amino acids fromFGF21 (or any FGF having low affinity for FGFR4, including FGF3, FGF5,FGF7, FGF9 and FGF10), such as at least 1, at least 2, at least 3, atleast 4, at least 5, at least 10, or at least 15, such as 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 correspondingamino acids from FGF21 (e.g., see SEQ ID NOS: 21, 219, 221, 222 and223). In some examples, the mutant FGF1 protein is a mutated version ofthe mature protein (e.g., SEQ ID NO: 5), such as one containing at least1, at least 2, at least 3, at least 4, at least 5, at least 6, at least7, at least 8, at least 9 or at least 10 amino acid substitutions (suchas 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40 or 41 substitutions), such as one or more of those shown inTable 1. In some examples, the mutant FGF1 protein includes both anN-terminal truncation and point mutations. In some examples, the mutantFGF1 protein includes at least 120 consecutive amino acids from aminoacids 5-141 of FGF1 (e.g., of SEQ ID NO: 2 or 4), (which in someexamples can include 1-20 point mutations, such as substitutions,deletions, or additions).

In some examples, the FGF1 mutants provided herein are used to generatea chimeric protein, such as an FGF1/FGF21, FGF1/FGF19,FGF1/β-Klotho-binding protein, FGF1/FGFR1c-binding protein orFGF1/β-Klotho-binding protein/FGFR1c-binding protein. For example, theC-terminal end or the N-terminal end of the disclosed FGF1 mutants canbe joined directly or indirectly to the N-terminal end of a C-terminalfragment of FGF21 or FGF19, such as SEQ ID NO: 86 or 100, respectively.Similarly, the C-terminal end of the disclosed FGF1 mutants can bejoined directly or indirectly to the N-terminal end of a β-Klothobinding domain (such as SEQ ID NO: 121, 122, 123, 124, 125, 126, 127,128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, 145, 146 or β-Klotho binding portion of SEQ ID NO: 168,169, 170 or 171), or the N-terminal end of the disclosed FGF1 mutantscan be joined directly or indirectly to the C-terminal end of aβ-Klotho-binding domain. In addition, the C-terminal end of thedisclosed FGF1 mutants can be joined directly or indirectly to theN-terminal end of a FGFR1c-binding domain (such as SEQ ID NO: 147, 148,149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162,163, 164, 165, 166, 167 or 190), or the N-terminal end of the disclosedFGF1 mutants can be joined directly or indirectly to the C-terminal endof a FGFR1c-binding domain. In some examples, the C-terminal end of thedisclosed FGF1 mutants can be joined directly or indirectly to anFGFR1c-binding domain (such as any of SEQ ID NOS: 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,165, 166, 167, 190 or FGFR1c-binding portion of 168, 169, 170 or 171)and a β-Klotho-binding domain, the N-terminal end of the disclosed FGF1mutants can be joined directly or indirectly to the C-terminal end of aFGFR1c-binding domain and a β-Klotho-binding domain, or both (such asSEQ ID NO: 168, 169, 170 or 171). Such chimeric proteins can be used toreduce blood glucose in a mammal, for example to treat a metabolicdisease.

Specific exemplary FGF1 mutant proteins are shown in SEQ ID NOS: 6, 7,8, 9, 10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 113,114, 115, 116, 117, 118, 119, 120, 191, 192, 193, 194, 195, 196, 197,198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211,212, 213, 214, 215, 216, 217, 218, 225, 226, 227, 228, 229, 230, 231,232, 233, 234, 235, 236, 237 and 238, which can be used to generate anyof the chimeras provided herein. Specific exemplary FGF1/FGF21 chimerasare shown in SEQ ID NOS: 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,219, 221, 222, and 223. Specific exemplary FGF1/FGF19 chimeras are shownin SEQ ID NOS: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,112, 220, and 224. Specific exemplary FGF1/β-Klotho-binding chimeras areshown in FIGS. 23-25 (and in SEQ ID NOS: 173, 174, 175, 177, 178, 179,181, 182, 183, 185, 186, and 187). Specific exemplaryFGF1/FGFR1c-binding chimeras are shown in FIGS. 23J and 241 (and in SEQID NOS: 188 and 189). Specific exemplary β-Klotho-binding/FGFR1c-bindingchimeras that can be linked directly or indirectly to an N- orC-terminal end of a FGF1 mutant protein are shown in SEQ ID NOS: 168,169, 170 and 171.

Also provided are FGFR1c-binding protein dimers and multimers (such astrimers) and their use to treat metabolic disease. An example is shownin FIG. 25E (also see SEQ ID NO: 190).

Also provided are isolated nucleic acid molecules encoding the disclosedmutant FGF1 proteins (which includes chimeras), and the FGFR1c bindingproteins. Vectors and cells that include such nucleic acid molecules arealso provided.

Methods of using the disclosed mutant FGF1 proteins and FGFR1c bindingprotein multimers (or nucleic acid molecules encoding such) areprovided, such as a mutated mature FGF1 protein having a deletion of atleast six contiguous N-terminal amino acids, at least one pointmutation, or combinations thereof, for example to reduce or eliminatemitogenic activity. In some examples the methods include administering atherapeutically effective amount of a disclosed mutant FGF1 protein orFGFR1c binding protein (or nucleic acid molecules encoding such) toreduce blood glucose in a mammal, such as a decrease of at least 5%. Insome examples the methods include administering a therapeuticallyeffective amount of a disclosed mutant FGF1 protein or FGFR1c bindingprotein multimer (or nucleic acid molecules encoding such) to treat ametabolic disease in a mammal. Exemplary metabolic diseases that can betreated with the disclosed methods include but are not limited to:diabetes (such as type 2 diabetes, non-type 2 diabetes, type 1 diabetes,latent autoimmune diabetes (LAD), or maturity onset diabetes of theyoung (MODY)), polycystic ovary syndrome (PCOS), metabolic syndrome(MetS), obesity, non-alcoholic steatohepatitis (NASH), non-alcoholicfatty liver disease (NAFLD), dyslipidemia (e.g., hyperlipidemia), andcardiovascular diseases (e.g., hypertension). In some examples, one ormore of these diseases are treated simultaneously with the disclosedFGF21 mutants. Also provided are methods of reducing fed and fastingblood glucose, improving insulin sensitivity and glucose tolerance,reducing systemic chronic inflammation, ameliorating hepatic steatosisin a mammal, reducing food intake, or combinations thereof, byadministering a therapeutically effective amount of a disclosed mutantFGF1 protein or FGFR1c binding protein multimer (or nucleic acidmolecules encoding such).

The foregoing and other objects and features of the disclosure willbecome more apparent from the following detailed description, whichproceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an exemplary wild-type mature FGF1 sequence (SEQ ID NO: 5),N-terminal deletions that can be made to mature FGF1 (SEQ ID NOS: 7, 8and 9), point mutations that can be made to mature FGF1 (SEQ ID NOS: 10and 11), and mutations to the heparan binding domain of FGF1 (SEQ IDNOS: 12 and 13).

FIGS. 2A and 2B are graphs showing blood glucose levels of (A) ob/obmice treated with control vehicle (PBS, open symbols), rFGF1 (0.5 mg/kgsubcutaneous, filled symbols), or rFGF1^(ΔNT) (0.5 mg/kg subcutaneous,dashed line, n=8-12). This shows that the non-mitogenic FGF1 variantrFGF1^(ΔNT) (SEQ ID NO: 7) has equivalent efficacy as wild-type FGF1 inlowering blood glucose levels in ob/ob diabetic mice. (B) Diet inducedobese (DIO) mice treated with control vehicle (PBS, open bars), rFGF1(0.5 mg/kg subcutaneous, filled bars), or rFGF1^(ΔNT) (0.5 mg/kgsubcutaneous, striped bars) at indicated times (n=10). This shows thatthe non-mitogenic FGF1 variant rFGF1^(ΔNT) has equivalent efficacy aswild-type FGF1 in lowering blood glucose levels in high fat diet fedobese mice. Subcutaneous injections of vehicle (PBS) or rFGF1 (0.5mg/kg) were performed on ad lib fed mice. Values are means±SEM.Statistics by two-tailed t test. *P<0.05, **P<0.01.

FIG. 2C is a graph showing food intake in DIO mice after control vehicle(PBS, open bar), rFGF1 (0.5 mg/kg subcutaneous, filled bars), orrFGF1^(ΔNT) (0.5 mg/kg subcutaneous, striped bar) treatment (n=5).

FIGS. 3A-3D show how an exemplary wild-type mature FGF1 sequence (SEQ IDNO: 5) can be mutated to include mutations that increase thermostabilityof FGF1 (M1, M2 and M3 deletions, SEQ ID NOS: 22, 28, and 40,respectively), which can be combined with FGF1 N-terminal deletionsand/or point mutations (SEQ ID NOS: 23, 24, 25, 26, 27, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 and51).

FIGS. 4A-4B show additional FGF1 mutant sequences that can be generatedfrom an exemplary wild-type mature FGF1 sequence (SEQ ID NO: 5) toinclude N-terminal deletions and/or point mutations (SEQ ID NOS: 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, and 66).

FIGS. 5A-5B show additional FGF1 mutant sequences that can be generatedfrom an exemplary wild-type mature FGF1 sequence (SEQ ID NO: 5) toinclude N-terminal deletions and/or point mutations (SEQ ID NOS: 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, and 84).

FIGS. 6A-6B show FGF21 (SEQ ID NO: 20) and a C-terminal portion of FGF21(SEQ ID NO: 86) that binds to beta-klotho, and how they can be attachedto FGF1 mutants described herein to form FGF1/FGF21 chimeric proteins(SEQ ID NOS: 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, and 98). TheFGF1/FGF21 chimeras shown can further include one or more of K12V andN95V FGF1 non mitogenic mutations (or other mutations disclosed herein,such as those listed in Table 1) that have longer glucose loweringduration.

FIGS. 7A-7B show FGF19 (SEQ ID NO: 99) and a C-terminal portion of FGF19(SEQ ID NO: 100) that binds to beta-klotho, and how they can be attachedto FGF1 mutants described herein to form FGF1/FGF19 chimeric proteins(SEQ ID NOS: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, and112). The FGF1/FGF19 chimeras shown can further include one or more ofK12V and N95V FGF1 non mitogenic mutations that have longer glucoselowering duration.

FIG. 8 is a digital image showing the effect of intracellular signalingwith M1 thru M5 peptides (SEQ ID NOS: 22, 28, 40, 54 and 212,respectively). HEK293 cells were serum starved and then treated with theindicated peptides at 10 ng/ml concentration for 15 min. Total celllysates were subject to western blots with indicated antibodies.

FIG. 9 is a digital image showing peptides NT1 (SEQ ID NO: 7), NT2 (SEQID NO: 8), or NT3 (SEQ ID NO: 9) KN (SEQ ID NO: 10), and KLE (SEQ ID NO:11) and their ability to affect intracellular signaling. HEK293 cellswere serum starved and then treated with the indicated peptides at 10ng/ml concentration for 15 min. Total cell lysates were subject towestern blots with indicated antibodies.

FIG. 10 is a digital image showing peptides FGF1 (SEQ ID NO: 5) and NT1(SEQ ID NO: 7) and their ability to affect intracellular signaling.HEK293 cells were serum starved and then treated with the indicatedpeptides at 10 ng/ml concentration for 15 min. Total cell lysates weresubject to western blots with indicated antibodies.

FIGS. 11A and 11B are graphs showing the glucose lowering effects forM1, M2, and M3 in ob/ob mice. Mice were 5 mo old C57BL/6J ob/ob onnormal chow. The peptides were injected SQ (0.5 mg/kg).

FIG. 12 shows in vivo glucose lowering effects correlate with FGFRmediated signaling. Mice were 5 mo old C57BL/6J ob/ob on normal chow.The peptides NT1 (SEQ ID NO: 7) and NT2 (SEQ ID NO: 8), were injected SQ(0.5 mg/kg).

FIG. 13 is a digital image showing that the in vivo glucose loweringeffect correlate with FGFR mediated signaling. Serum starved HEK 293cells were treated with indicated peptides (10 ng/ml) for 15 min andsubject to western blot. FGF1^(ΔNTPPrep1) and FGF1^(ΔNTPrep2) are thesame sequence, just independent preparations of the protein.

FIG. 14 is a graph showing blood glucose levels 0 to 120 hrs followingadministration of a single injection of FGF1-KLE (SEQ ID NO: 11) orFGF1-KN (SEQ ID NO: 10). The FGF1-KN mutant retained the ability tolower glucose for 120 hrs while FGF1-KLE fails to lower glucose.

FIG. 15A compares the dose response of downstream FGFR signaling inducedby rFGF1 (SEQ ID NO: 5) and NT1 (rFGF1^(ΔNT), SEQ ID NO: 7). FIG. 15B isthe same as FIG. 2C. FIG. 15C compares the dose response of rFGF1 andNT1 in lowering glucose in ob/ob mice. A. Western blot showingintracellular signaling in serum starved HEK293 cells after a 15 mintreatment with the indicated concentrations of PBS (vehicle),rFGF1^(ΔNT), or rFGF1. B. Food intake in DIO mice during 24 hr periodafter injection of control vehicle (PBS, open bar), rFGF1 (0.5 mg/kgsubcutaneous, filled bars), or rFGF^(ΔNT) (0.5 mg/kg subcutaneous,striped bar, n=5). C. Dose response of glucose lowering effects ofsubcutaneously delivered rFGF1^(ΔNT) (striped bars) in comparison torFGF1 (filled bars) in 12 week old ob/ob mice (n=6-12). ***P<0.005.

FIG. 16 is a bar graph showing blood glucose levels 0 hr, 16 hrs, or 24hrs following administration of PBS, NT1 (FGF1^(ΔNT), SEQ ID NO: 7), NT2(FGF1^(ΔNT2), SEQ ID NO: 8), or NT3 (FGF1^(ΔNT3), SEQ ID NO: 9). Notethat if the N-terminus is truncated at 14 amino acids, glucose loweringability is substantially decreased (NT2). Mice were 5 mo old C57BL/6Job/ob on normal chow. The peptides were injected SQ (0.5 mg/kg).

FIGS. 17A and 17B are bar graphs showing that NT1 (SEQ ID NO: 7) failsto lower blood glucose levels in HFD-fed aP2-Cre; FGFR1 f/f mice (mutantFGFR1KO mice). Blood glucose levels in 8 month old HFD-fed wildtypeFGFR1 f/f (control open bars) or adipose-specific FGFR1 knockout(mutant, R1KO, aP2-Cre; FGFR1 f/f, filled bars) mice after NT1 treatment(murine rFGF1^(ΔNT), 0.5 mg/kg subcutaneous injection, n=5 per group).Values are means±SEM. (A) shows the raw blood glucose levels, (B) showsthe data normalized to initial blood glucose as 100%.

FIGS. 18A and 18B are bar graphs showing that mouse rFGF1 (amino acids1-15 of SEQ ID NO 4) fails to lower blood glucose levels in HFD-fedaP2-Cre; FGFR1 f/f mice (FGFR1 KO, filled bars). Blood glucose levels in8 month old HFD-fed wild type (FGFR1 f/f, black bars) oradipose-specific FGFR1 knockout (R1 KO, aP2-Cre; FGFR1 f/f, dotted bars)mice after rFGF1 treatment (murine rFGF1, 0.5 mg/kg subcutaneousinjection, n−=5 per group). Values are means±SEM. (A) shows the rawblood glucose levels, (B) shows the data normalized to initial bloodglucose as 100%. Murine FGF1 (amino acids 1-15 of SEQ ID NO 4) is ˜96%homologous to the human sequence (SEQ ID NO: 5)

FIG. 19 is a bar graph showing that FGF1 mutations K118E (SEQ ID NO: 12)and K118N (SEQ ID NO: 13) fail to lower blood glucose levels in DIOmice. Blood glucose levels in 7 months HFD-fed C57BL/6J mice after PBS(open bar), K118E (filled bar), and K118N (hatched bar) treatment (0.5mg/kg subcutaneous injection, n=4-8 per group). Values are means±SEM.

FIG. 20 shows a native FGF1 sequence (SEQ ID NO: 5) and eight heparanbinding mutant FGF1 KKK analogs (SEQ ID NOS: 113, 114, 115, 116, 117,118, 119, and 120).

FIGS. 21 and 22 show that FGF1 heparan binding mutant KKK lowersglucose.

FIGS. 23-26 show exemplary arrangements of FGF1 mutant/β-Klotho-bindingchimeras and FGFR1c-binding protein dimers. Exemplary sequences areshown in SEQ ID NOS: 172, 173, 174, 175, 176, 177, 178, 179, 180, 181,182, 183, 184, 185, 186, 187, 188, 189 and 190. Although monomers ordimers of FGFR1c- or β-Klotho-binding proteins are shown, in someexamples greater multimers are used, such as trimers, etc. In addition,the FGF1 mutant/β-Klotho-binding chimeras can be made into FGF1mutant/FGFR1c-binding chimeras by replacing the β-Klotho-binding portionwith an FGFR1c-binding portion (e.g., as shown in FIGS. 23J and 241 forΔNT FGF1). Furthermore, FGFR1c-binding portion(s) can be included in theFGF1 mutant/β-Klotho-binding chimeras (e.g., as shown in FIGS. 23K and24J for ΔNT FGF1). The sequence of CC2240 is shown in SEQ ID NO: 121 andC2987 in SEQ ID NO: 148.

FIG. 27 shows exemplary FGF1 mutant sequences that include an R35Esubstitution (SEQ ID NOS: 191-198).

FIG. 28 shows exemplary FGF1 mutant sequences that include an R35Vsubstitution (SEQ ID NOS: 199-206).

FIG. 29 shows exemplary FGF1 mutant sequences (SEQ ID NOS: 207-211).This free cysteine (C117) forms intermolecular disulfide bonds that leadto protein aggregation. The mutation to valine is designed to improvestability, hence it is introduced in combination with other pointmutations. KKKR are putative heparin binding residues. KY, KE, KEY, KNYare various combinations of point mutations to residues that interactwith the FGF receptors (K=K12, E=E87, Y=Y94, N=N95).

FIGS. 30A-30E show exemplary FGF1 mutant sequences that are mutated to(A) increase stability (SEQ ID NOS: 54, 212-218 and 113), (B) chimeras(SEQ ID NOS: 219-224), (C) increase stability and decrease mitogencity(SEQ ID NOS: 225-229, (D) increase stability and decrease mitogencity(SEQ ID NOS: 230-233), and (E) increase stability and decreasemitogencity (SEQ ID NOS:234-238).

FIG. 31 shows an alignment of FGF1 (SEQ ID NO: 5) and FGF2 (SEQ ID NO:85), with amino acids that form beta strands in bold, and other relevantresidues highlighted and their interaction noted.

FIGS. 32A-32D are graphs showing the affect of FGF1 mutations on bloodglucose lowering and feeding effects in vivo. Peptides KN (Salk_(—)004,SEQ ID NO: 10), KKK (Salk_(—)010, SEQ ID NO: 226), FGF1 (SEQ ID NO: 5),KLE (Salk_(—)011, SEQ ID NO: 11), FGF1^(ΔNT) (NT1) (SEQ ID NO: 7) andFGF1^(ΔNTKN) (Salk_(—)009, SEQ ID NO: 225) were tested.

FIGS. 33A-33B are bar graphs showing the affect of FGF1 mutations on (A)blood glucose lowering and (B) feeding effects in vivo. Peptides FGF1(SEQ ID NO: 5), Salk_(—)013 (SEQ ID NO: 31), and Salk_(—)012 (SEQ ID NO:79) were tested.

FIGS. 34A-34B are bar graphs showing the affect of FGF1 mutations on (A)blood glucose lowering and (B) feeding effects in vivo. PeptidesSalk_(—)014 (SEQ ID NO: 230), Salk_(—)024 (SEQ ID NO: 84), Salk_(—)025(SEQ ID NO: 208), Salk_(—)026 (SEQ ID NO: 209), and Salk_(—)023 (SEQ IDNO: 38) were tested.

FIGS. 35A-35B are bar graphs showing the affect of FGF1 mutations on (A)blood glucose lowering and (B) feeding effects in vivo. PeptidesSalk_(—)014 (SEQ ID NO: 230), Salk_(—)024 (SEQ ID NO: 84), Salk_(—)025(SEQ ID NO: 208), and Salk_(—)026 (SEQ ID NO: 209), and Salk_(—)023 (SEQID NO: 38) were tested.

FIGS. 36A-36B are bar graphs showing the affect of FGF1 mutations on (A)blood glucose lowering and (B) feeding effects in vivo. PeptidesSalk_(—)014 (SEQ ID NO: 230) and Salk_(—)032 (SEQ ID NO: 215) weretested.

FIGS. 37A-37B are bar graphs showing the affect of a FGF1-FGF19 chimeraon (A) blood glucose lowering and (B) feeding effects in vivo. PeptidesSalk_(—)014 (SEQ ID NO: 230) and Salk_(—)019 (SEQ ID NO: 224) weretested.

FIG. 38 is a bar graph showing the affect of a FGF1-FGF21 chimera on (A)blood glucose lowering and (B) feeding effects in vivo. Peptides FGF1(SEQ ID NO: 5), FGF1^(ΔNT) (SEQ ID NO: 7), FGF21 (SEQ ID 20) andFGF1-FGF21 chimera (SEQ ID NO: 114+SEQ ID NO: 86) were tested.

SEQUENCE LISTING

The nucleic and amino acid sequences are shown using standard letterabbreviations for nucleotide bases, and three letter code for aminoacids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleicacid sequence is shown, but the complementary strand is understood asincluded by any reference to the displayed strand.

SEQ ID NOS: 1 and 2 provide an exemplary human FGF1 nucleic acid andprotein sequences, respectively. Source: GenBank Accession Nos:BC032697.1 and AAH32697.1. Heparan binding residues are amino acids127-129 and 133-134.

SEQ ID NOS: 3 and 4 provide an exemplary mouse FGF1 nucleic acid andprotein sequences, respectively. Source: GenBank Accession Nos:BC037601.1 and AAH37601.1.

SEQ ID NO: 5 provides an exemplary mature form of FGF1 (140 aa,sometimes referred to in the art as FGF1 15-154)

SEQ ID NO: 6 provides an exemplary mature form of FGF1 with anN-terminal deletion.

SEQ ID NO: 7 provides an exemplary mature form of FGF1 with anN-terminal deletion (FGF1^(ΔNT)(10-140αα)).

SEQ ID NO: 8 provides an exemplary mature form of FGF1 with anN-terminal deletion (FGF1^(ΔNT2)(14-140αα)).

SEQ ID NO: 9 provides an exemplary mature form of FGF1 with anN-terminal deletion (FGF1^(ΔNT3)(12-140αα)).

SEQ ID NO: 10 provides an exemplary mature form of FGF1 with pointmutations (K12V, N95V, wherein numbering refers to SEQ ID NO: 5) toreduce mitogenic activity.

SEQ ID NO: 11 provides an exemplary mature form of FGF1 with pointmutations (K12V, L46V, E87V, N95V, P134V, wherein numbering refers toSEQ ID NO: 5) to reduce mitogenic activity.

SEQ ID NOS: 12 and 13 provide exemplary mature forms of FGF1 withmutations in the heparan binding domain (K118N or K118E, respectively,wherein numbering refers to SEQ ID NO: 5). In some examples thesesequences further include MFNLPPG at their N-terminus. Such proteinshave reduced mitogenicity as compared to wild-type FGF 1.

SEQ ID NOS: 14-17 provide exemplary mutated FGF1 nuclear exportsequences.

SEQ ID NO: 18 provides a coding sequence for SEQ ID NO: 6.

SEQ ID NOS: 19 and 20 provide an exemplary human FGF21 nucleic acid andprotein sequence. Obtained from GenBank Accession Nos. AY359086 andAAQ89444.1. The mature form of FGF21 is about amino acids 21-208.

SEQ ID NO: 21 provides an exemplary N-terminally truncated form of FGF1,wherein the four N-terminal amino acids are from FGF21 (amino acids40-43 of SEQ ID NO: 20).

SEQ ID NO: 22 provides an exemplary mature form of FGF1 with pointmutations (K12V, C117V and P134V wherein numbering refers to SEQ ID NO:5) to reduce mitogenic activity and increase thermostability. From Xiaet al., PLoS One. 7(11):e48210, 2012.

SEQ ID NO: 23 (FGF1(1-140αα)M1a) provides an exemplary mature form ofFGF1 with point mutations (K12V, N95V, C117V, and P134V whereinnumbering refers to SEQ ID NO: 5) to reduce mitogenic activity andincrease thermostability.

SEQ ID NO: 24 (FGF1^(ΔNT1) (1-140αα)M1) provides an exemplaryN-terminally truncated form of FGF1 with point mutations (K12V, C117V,and P134V wherein numbering refers to SEQ ID NO: 5) to reduce mitogenicactivity and increase thermostability.

SEQ ID NO: 25 (FGF1^(ΔNT3) (1-140αα)M1a) provides an exemplaryN-terminally truncated form of FGF1 with point mutations (K12V, C117V,and P134V wherein numbering refers to SEQ ID NO: 5) to reduce mitogenicactivity and increase thermostability.

SEQ ID NO: 26 (FGF1^(ΔNT1) (1-140αα)M1a) provides an exemplaryN-terminally truncated form of FGF1 with point mutations (K12V, N95V,C117V, and P134V wherein numbering refers to SEQ ID NO: 5) to reducemitogenic activity, and increase thermostability.

SEQ ID NO: 27 (FGF1^(ΔNT3) (1-140αα)M1a) provides an exemplaryN-terminally truncated form of FGF1 with point mutations (K12V, N95V,C117V, and P134V wherein numbering refers to SEQ ID NO: 5) to reducemitogenic activity, and increase thermostability

SEQ ID NO: 28 (FGF1(1-140αα)M2) provides an exemplary mature form ofFGF1 with point mutations (L44F, C83T, C117V, and F132W whereinnumbering refers to SEQ ID NO: 5) to reduce mitogenic activity andincrease thermostability. From Xia et al., PLoS One. 7(11):e48210, 2012.

SEQ ID NO: 29 (FGF1(1-140αα)M2a) provides an exemplary mature form ofFGF1 with point mutations (L44F, C83T, N95V, C117V, and F132W whereinnumbering refers to SEQ ID NO: 5) to reduce mitogenic activity andincrease thermostability.

SEQ ID NO: 30 (FGF1(1-140αα)M2b) provides an exemplary mature form ofFGF1 with point mutations (K12V, L44F, C83T, C117V, and F132W whereinnumbering refers to SEQ ID NO: 5) to reduce mitogenic activity andincrease thermostability.

SEQ ID NO: 31 (FGF1(1-140αα)M2c) provides an exemplary mature form ofFGF1 with point mutations (K12V, L44F, C83T, N95V, C117V, and F132Wwherein numbering refers to SEQ ID NO: 5) to reduce mitogenic activityand increase thermostability.

SEQ ID NO: 32 (FGF1^(ΔNT1)(10-140αα)M2) provides an exemplaryN-terminally truncated form of FGF1 with point mutations (L44F, C83T,C117V, and F132W wherein numbering refers to SEQ ID NO: 5) to reducemitogenic activity and increase thermostability.

SEQ ID NO: 33 (FGF1^(ΔNT3)(12-140αα)M2) provides an exemplaryN-terminally truncated form of FGF1 with point mutations (L44F, C83T,C117V, and F132W wherein numbering refers to SEQ ID NO: 5) to reducemitogenic activity and increase thermostability.

SEQ ID NO: 34 (FGF1^(ΔNT1)(10-140αα)M2a) provides an exemplaryN-terminally truncated form of FGF1 with point mutations (L44F, C83T,N95V, C117V, and F132W wherein numbering refers to SEQ ID NO: 5) toreduce mitogenic activity and increase thermostability.

SEQ ID NO: 35 (FGF1^(ΔNT3)(12-140αα)M2a) provides an exemplaryN-terminally truncated form of FGF1 with point mutations (L44F, C83T,N95V, C117V, and F132W wherein numbering refers to SEQ ID NO: 5) toreduce mitogenic activity and increase thermostability.

SEQ ID NO: 36 (FGF1^(ΔNT1)(10-140αα)M2b) provides an exemplaryN-terminally truncated form of FGF1 with point mutations (K12V, L44F,C83T, C117V, and F132W wherein numbering refers to SEQ ID NO: 5) toreduce mitogenic activity and increase thermostability.

SEQ ID NO: 37 (FGF1^(ΔNT3)(12-140αα)M2b) provides an exemplaryN-terminally truncated form of FGF1 with point mutations (K12V, L44F,C83T, C117V, and F132W wherein numbering refers to SEQ ID NO: 5) toreduce mitogenic activity and increase thermostability.

SEQ ID NO: 38 (FGF1^(ΔNT1)(10-140αα)M2c) provides an exemplaryN-terminally truncated form of FGF1 with point mutations (K12V, L44F,C83T, N95V, and C117V, F132W wherein numbering refers to SEQ ID NO: 5)to reduce mitogenic activity and increase thermostability.

SEQ ID NO: 39 (FGF1^(ΔNT3)(12-140αα)M2c) provides an exemplaryN-terminally truncated form of FGF1 with point mutations (K12V, L44F,C83T, N95V, and C117V, F132W wherein numbering refers to SEQ ID NO: 5)to reduce mitogenic activity and increase thermostability.

SEQ ID NO: 40 (FGF1(1-140αα)M3) provides an exemplary mature form ofFGF1 with mutations (L44F, M67I, L73V, V109L, L111I, C117V, A103G, R119GΔ104-106, and Δ120-122, wherein numbering refers to SEQ ID NO: 5) toreduce mitogenic activity and increase thermostability. From Xia et al.,PLoS One. 7(11):e48210, 2012.

SEQ ID NO: 41 (FGF1(1-140αα)M3a) provides an exemplary mature form ofFGF1 with mutations (K12V, L44F, M67I, L73V, V109L, L111I, C117V, A103G,R119G, Δ104-106, and Δ120-122 wherein numbering refers to SEQ ID NO: 5)to reduce mitogenic activity and increase thermostability.

SEQ ID NO: 42 (FGF1(1-140αα)M3b) provides an exemplary mature form ofFGF1 with mutations (K12V, L44F, M67I, L73V, N95V, V109L, L111I, C117V,A103G, R119G, Δ104-106, and Δ120-122 wherein numbering refers to SEQ IDNO: 5) to reduce mitogenic activity and increase thermostability.

SEQ ID NO: 43 (FGF1(1-140αα)M3c) provides an exemplary mature form ofFGF1 with mutations (K12V, L44F, M67I, L73V, N95V, V109L, L111I, C117V,A103G, R119G, Δ104-106, and Δ120-122 wherein numbering refers to SEQ IDNO: 5) to reduce mitogenic activity and increase thermostability.

SEQ ID NO: 44 (FGF1^(ΔNT1) (1-140αα)M3) provides an exemplaryN-terminally truncated form of FGF1 with mutations (L44F, M67I, L73V,V109L, L111I, C117V, A103G, R119G, Δ104-106, and Δ120-122 whereinnumbering refers to SEQ ID NO: 5) to reduce mitogenic activity andincrease thermostability.

SEQ ID NO: 45 (FGF1^(ΔNT3) (1-140αα)M3) provides an exemplaryN-terminally truncated form of FGF1 with mutations (L44F, M67I, L73V,V109L, L111I, C117V, A103G, R119G, Δ104-106, and Δ120-122 whereinnumbering refers to SEQ ID NO: 5) to reduce mitogenic activity andincrease thermostability.

SEQ ID NO: 46 (FGF1^(ΔNT1) (1-140αα)M3a) provides an exemplaryN-terminally truncated form of FGF1 with mutations (K12V, L44F, M67I,L73V, V109L, L111I, C117V, A103G, R119G, Δ104-106, and Δ120-122 whereinnumbering refers to SEQ ID NO: 5) to reduce mitogenic activity andincrease thermostability.

SEQ ID NO: 47 (FGF1^(ΔNT3) (1-140αα)M3a) provides an exemplaryN-terminally truncated form of FGF1 with mutations (K12V, L44F, M67I,L73V, V109L, L111I, C117V, A103G, R119G, Δ104-106, and Δ120-122 whereinnumbering refers to SEQ ID NO: 5) to reduce mitogenic activity andincrease thermostability.

SEQ ID NO: 48 (FGF1^(ΔNT1) (1-140αα)M3b) provides an exemplaryN-terminally truncated form of FGF1 with mutations (L44F, M67I, L73V,N95V, V109L, L111I, C117V, A103G, R119G, Δ104-106, and Δ120-122 whereinnumbering refers to SEQ ID NO: 5) to reduce mitogenic activity andincrease thermostability.

SEQ ID NO: 49 (FGF1^(ΔNT3) (1-140αα)M3b) provides an exemplaryN-terminally truncated form of FGF1 with mutations (L44F, M67I, L73V,N95V, V109L, L111I, C117V, A103G, R119G, Δ104-106, and Δ120-122 whereinnumbering refers to SEQ ID NO: 5) to reduce mitogenic activity andincrease thermostability.

SEQ ID NO: 50 (FGF1^(ΔNT1) (1-140αα)M3c) provides an exemplaryN-terminally truncated form of FGF1 with mutations (K12V, L44F, M67I,L73V, N95V, V109L, L111I, C117V, A103G, R119G, Δ104-106, and Δ120-122wherein numbering refers to SEQ ID NO: 5) to reduce mitogenic activityand increase thermostability.

SEQ ID NO: 51 (FGF1^(ΔNT3) (1-140αα)M3c) provides an exemplaryN-terminally truncated form of FGF1 with point mutations (K12V, L44F,M67I, L73V, N95V, V109L, L111I, C117V, A103G, R119G, Δ104-106, andΔ120-122 wherein numbering refers to SEQ ID NO: 5) to reduce mitogenicactivity and increase thermostability.

SEQ ID NO: 52 (FGF1 (1-140αα) provides an exemplary mature form of FGF1with point mutations (K12V, N95V, and K118N wherein numbering refers toSEQ ID NO: 5).

SEQ ID NO: 53 (FGF1 (1-140αα) provides an exemplary mature form of FGF1with point mutations (K12V, N95, and K118E wherein numbering refers toSEQ ID NO: 5).

SEQ ID NO: 54 FGF1 (1-140αα) K12V, N95V, C117V provides an exemplarymature form of FGF1 with point mutations (K12V, N95V, and C117V whereinnumbering refers to SEQ ID NO: 5).

SEQ ID NO: 55 (FGF1 (1-140αα) provides an exemplary mature form of FGF1with point mutations (K12V, N95V, C117V, and K118N wherein numberingrefers to SEQ ID NO: 5).

SEQ ID NO: 56 (FGF1 (1-140αα) provides an exemplary mature form of FGF1with point mutations (K12V, N95V, C117V, and K118E wherein numberingrefers to SEQ ID NO: 5).

SEQ ID NO: 57 (FGF1^(ΔNT) (10-140αα) provides an exemplary N-terminallytruncated FGF1 with point mutations (K12V and N95V, wherein numberingrefers to SEQ ID NO: 5).

SEQ ID NO: 58 (FGF1^(ΔNT2) (12-140αα) provides an exemplary N-terminallytruncated FGF1 with point mutations (K12V, and N95V, wherein numberingrefers to SEQ ID NO: 5).

SEQ ID NO: 59 (FGF1^(ΔNT) (10-140αα) provides an exemplary N-terminallytruncated FGF1 with a point mutation (K12V, wherein numbering refers toSEQ ID NO: 5).

SEQ ID NO: 60 (FGF1^(ΔNT2) (12-140αα) provides an exemplary N-terminallytruncated FGF1 with a point mutation (K12V, wherein numbering refers toSEQ ID NO: 5).

SEQ ID NO: 61 (FGF1^(ΔNT) (10-140αα) provides an exemplary N-terminallytruncated FGF1 with a point mutation (N95V, wherein numbering refers toSEQ ID NO: 5).

SEQ ID NO: 62 (FGF1^(ΔNT2) (12-140αα) provides an exemplary N-terminallytruncated FGF1 with a point mutation (N95V, wherein numbering refers toSEQ ID NO: 5).

SEQ ID NO: 63 (FGF1^(ΔNT) (10-140αα) provides an exemplary N-terminallytruncated FGF1 with point mutations (K12V, N95V, and K118N, whereinnumbering refers to SEQ ID NO: 5).

SEQ ID NO: 64 (FGF1^(ΔNT2) (12-140αα) provides an exemplary N-terminallytruncated FGF1 with point mutations (K12V, N95V, and K118E, whereinnumbering refers to SEQ ID NO: 5).

SEQ ID NO: 65 (FGF1^(ΔNT) (10-140αα) provides an exemplary N-terminallytruncated FGF1 with a point mutation (K118N, wherein numbering refers toSEQ ID NO: 5).

SEQ ID NO: 66 (FGF1^(ΔNT2) (12-140αα) provides an exemplary N-terminallytruncated FGF1 with a point mutation (K118E, wherein numbering refers toSEQ ID NO: 5).

SEQ ID NO: 67 (FGF1 (1-140αα) provides an exemplary mature form of FGF1with point mutations (K9T and N10T wherein numbering refers to SEQ IDNO: 5).

SEQ ID NO: 68 (FGF1 (1-140αα) provides an exemplary mature form of FGF1with point mutations (K9T, N10T, and N95V, wherein numbering refers toSEQ ID NO: 5).

SEQ ID NO: 69 (FGF1 (1-140αα) provides an exemplary mature form of FGF1with point mutations (K9T, N10T, and K118N, wherein numbering refers toSEQ ID NO: 5).

SEQ ID NO: 70 (FGF1 (1-140αα) provides an exemplary mature form of FGF1with a mutant NLS sequence.

SEQ ID NO: 71 (FGF1^(ΔNT) (1-140αα) provides an exemplary N-terminallytruncated form of FGF1 with point mutations (Q40P and S471, whereinnumbering refers to SEQ ID NO: 5).

SEQ ID NO: 72 (FGF1^(ΔNT3) (1-140αα) provides an exemplary N-terminallytruncated form of FGF1 with point mutations (Q40P and S471, whereinnumbering refers to SEQ ID NO: 5).

SEQ ID NO: 73 (FGF1 (1-140αα) provides an exemplary mature form of FGF1with point mutations (K12V, Q40P, S47I, and N95V wherein numberingrefers to SEQ ID NO: 5).

SEQ ID NO: 74 FGF1^(ΔNT) (1-140αα) provides an exemplary N-terminallytruncated form of FGF1 with point mutations (K12V, Q40P, S47I, and N95V,wherein numbering refers to SEQ ID NO: 5).

SEQ ID NO: 75 (FGF1^(ΔNT3) (1-140αα) provides an exemplary N-terminallytruncated form of FGF1 with point mutations (K12V, Q40P, S47I, and N95V,wherein numbering refers to SEQ ID NO: 5).

SEQ ID NO: 76 (FGF1^(ΔNT) (1-140αα) provides an exemplary N-terminallytruncated form of FGF1 with point mutations (Q40P, S47I, and H93G,wherein numbering refers to SEQ ID NO: 5).

SEQ ID NO: 77 (FGF1^(ΔNT3) (1-140αα) provides an exemplary N-terminallytruncated form of FGF1 with point mutations (Q40P, S47I, and H93G,wherein numbering refers to SEQ ID NO: 5).

SEQ ID NO: 78 (FGF1 (1-140αα) provides an exemplary mature form of FGF1with point mutations (K12V, Q40P, S47I, H93G, and N95V, whereinnumbering refers to SEQ ID NO: 5).

SEQ ID NO: 79 (FGF1^(ΔNT) (1-140αα) provides an exemplary N-terminallytruncated form of FGF1 with point mutations (K12V, Q40P, S47I, H93G, andN95V, wherein numbering refers to SEQ ID NO: 5).

SEQ ID NO: 80 (FGF1^(ΔNT3) (1-140αα) provides an exemplary N-terminallytruncated form of FGF1 with point mutations (K12V, Q40P, S47I, H93G, andN95V, wherein numbering refers to SEQ ID NO: 5).

SEQ ID NO: 81 (FGF1^(ΔNT) (1-140αα) provides an exemplary N-terminallytruncated form of FGF1 with point mutations (C117P and K118V, whereinnumbering refers to SEQ ID NO: 5).

SEQ ID NO: 82 (FGF1^(ΔNT3) (1-140αα) provides an exemplary N-terminallytruncated form of FGF1 with point mutations (C117P and K118V, whereinnumbering refers to SEQ ID NO: 5).

SEQ ID NO: 83 (FGF1 (1-140αα) provides an exemplary mature form of FGF1with point mutations (K12V, N95V, C117P, and K118V, wherein numberingrefers to SEQ ID NO: 5).

SEQ ID NO: 84 (FGF1 (1-140αα) provides an exemplary mature form of FGF1with a point mutation (R35E, wherein numbering refers to SEQ ID NO: 5).Such an antagonist can be used to treat hypoglycemia or type I diabetes.

SEQ ID NO: 85 provides an exemplary portion of an FGF2 protein sequence.

SEQ ID NO: 86 provides an exemplary C-terminal FGF21 protein sequence(P¹⁶⁸-S²⁰⁹hFGF21^(C-tail)). This fragment can be attached at itsN-terminus to the C-terminus of any FGF1 mutant provided herein togenerate an FGF1/FGF21 chimera.

SEQ ID NO: 87 provides an exemplary FGF1/FGF21 chimera, which containsthe K12V and N95V FGF1 point mutations. The FGF21 portion is amino acids136 to 177.

SEQ ID NO: 88 provides an exemplary FGF1/FGF21 chimera(FGF1^(ΔNT)-FGF21^(C-tail)). The FGF21 portion is amino acids 127 to168.

SEQ ID NO: 89 provides an exemplary FGF1/FGF21 chimera(FGF1^(ΔNT3)-FGF21^(C-tail)) The FGF21 portion is amino acids 125 to166.

SEQ ID NO: 90 provides an exemplary FGF1/FGF21 chimera(M1-FGF21^(C-tail)). The FGF1 portion includes point mutations K¹²V,C¹¹⁷V, and P¹³⁴V. The FGF21 portion is amino acids 127 to 168.

SEQ ID NO: 91 provides an exemplary FGF1/FGF21 chimera(M1-FGF21^(C-tail)). The FGF1 portion includes point mutations K¹²V,C¹¹⁷V, and P¹³⁴V. The FGF21 portion is amino acids 125 to 166.

SEQ ID NO: 92 provides an exemplary FGF1/FGF21 chimera(M1-FGF21^(C-tail)). The FGF1 portion includes point mutations K¹²V,C¹¹⁷V, and P¹³⁴V. The FGF21 portion is amino acids 136 to 177.

SEQ ID NO: 93 provides an exemplary FGF1/FGF21 chimera(M2-FGF21^(C-tail)). The FGF1 portion includes point mutations L⁴⁴F,C⁸³T, C¹¹⁷V, and F¹³²W. The FGF21 portion is amino acids 127 to 168.

SEQ ID NO: 94 provides an exemplary FGF1/FGF21 chimera(M2-FGF21^(C-tail)). The FGF1 portion includes point mutations L⁴⁴F,C⁸³T, C¹¹⁷V, and F¹³²W. The FGF21 portion is amino acids 125 to 166.

SEQ ID NO: 95 provides an exemplary FGF1/FGF21 chimera(M2-FGF21^(C-tail)). The FGF1 portion includes point mutations L⁴⁴F,C⁸³T, C¹¹⁷V, and F¹³²W. The FGF21 portion is amino acids 136 to 177.

SEQ ID NO: 96 provides an exemplary FGF1/FGF21 chimera(M3-FGF21^(C-tail)). The FGF1 portion includes mutations L⁴⁴F, M⁶⁷I,L⁷³V, V109L, L^(III)I, C¹¹⁷V, A¹⁰³G, R¹¹⁹G, Δ¹⁰⁴⁻¹⁰⁶ and Δ¹²⁰⁻¹²². TheFGF21 portion is amino acids 121 to 162.

SEQ ID NO: 97 provides an exemplary FGF1/FGF21 chimera(M3-FGF21^(C-tail)). The FGF1 portion includes mutations L⁴⁴F, M⁶⁷I,L⁷³V, V¹⁰⁹L, L¹¹¹I, C¹¹⁷V, A¹⁰³G, R¹¹⁹G, Δ¹⁰⁴⁻¹⁰⁶ and Δ¹²⁰-122. TheFGF21 portion is amino acids 119 to 160.

SEQ ID NO: 98 provides an exemplary FGF1/FGF21 chimera(M3-FGF21^(C-tail)). The FGF1 portion includes mutations L⁴⁴F, M⁶⁷I,L⁷³V, V¹⁰⁹L, L¹¹¹I, C¹¹⁷V, A¹⁰³G, R¹¹⁹G, Δ¹⁰⁴⁻¹⁰⁶ and Δ¹²⁰⁻¹²². TheFGF21 portion is amino acids 130 to 171.

SEQ ID NO: 99 provides an exemplary FGF19 protein sequence. The matureform of FGF19 is amino acids 23 to 216.

SEQ ID NO: 100 provides an exemplary C-terminal FGF19 protein sequence(L¹⁶⁹-K²¹⁶ h FGF19C-tail). This fragment can be attached at itsN-terminus to the C-terminus of any FGF1 mutant provided herein togenerate an FGF1/FGF19 chimera.

SEQ ID NO: 101 provides an exemplary FGF1/FGF19 chimera. The FGF1portion includes point mutations K¹²V, and N⁹⁵V. The FGF19 portion isamino acids 136 to 183.

SEQ ID NO: 102 provides an exemplary FGF1/FGF19 chimera(FGF1^(ΔNT)-FGF19^(C-tail)). The FGF19 portion is amino acids 127 to174.

SEQ ID NO: 103 provides an exemplary FGF1/FGF19 chimera(FGF1^(ΔNT3)-FGF19^(C-tail)). The FGF19 portion is amino acids 125 to172.

SEQ ID NO: 104 provides an exemplary FGF1/FGF19 chimera(M1-FGF19^(C-tail)). The FGF1 portion includes point mutations K¹²V,C¹¹⁷V, and P¹³⁴V. The FGF19 portion is amino acids 136 to 183.

SEQ ID NO: 105 provides an exemplary FGF1/FGF19 chimera(M1-FGF19^(C-tail)). The FGF1 portion includes point mutations K¹²V,C¹¹⁷V, and P¹³⁴V. The FGF19 portion is amino acids 127 to 174.

SEQ ID NO: 106 provides an exemplary FGF1/FGF19 chimera(M1-FGF19^(C-tail)). The FGF1 portion includes point mutations K¹²V,C¹¹⁷V, and P¹³⁴V. The FGF19 portion is amino acids 125 to 172.

SEQ ID NO: 107 provides an exemplary FGF1/FGF19 chimera(M2-FGF19^(C-tail)). The FGF1 portion includes point mutations L⁴⁴F,C⁸³T, C¹¹⁷V, and F¹³²W. The FGF19 portion is amino acids 136 to 183.

SEQ ID NO: 108 provides an exemplary FGF1/FGF19 chimera(M2-FGF19^(C-tail)). The FGF1 portion includes point mutations L⁴⁴F,C⁸³T, C¹¹⁷V, and F¹³²W. The FGF19 portion is amino acids 127 to 174.

SEQ ID NO: 109 provides an exemplary FGF1/FGF19 chimera(M2-FGF19^(C-tail)). The FGF1 portion includes point mutations L⁴⁴F,C⁸³T, C¹¹⁷V, and F¹³²W. The FGF19 portion is amino acids 125 to 172.

SEQ ID NO: 110 provides an exemplary FGF1/FGF19 chimera(M3-FGF19^(C-tail)). The FGF1 portion includes mutations L⁴⁴F, M⁶⁷I,L⁷³V, V¹⁰⁹L, L¹¹¹I, C¹¹⁷V, A¹⁰³G, R¹¹⁹G, Δ¹⁰⁴⁻¹⁰⁶ and Δ¹²⁰⁻¹²². TheFGF19 portion is amino acids 130 to 177.

SEQ ID NO: 111 provides an exemplary FGF1/FGF19 chimera(M3-FGF19^(C-tail)). The FGF1 portion includes mutations L⁴⁴F, M⁶⁷I,L⁷³V, V¹⁰⁹L, L¹¹¹I, C¹¹⁷V, A¹⁰³G, R¹¹⁹G, Δ¹⁰⁴⁻¹⁰⁶ and Δ¹²⁰⁻¹²². TheFGF19 portion is amino acids 121 to 168.

SEQ ID NO: 112 provides an exemplary FGF1/FGF19 chimera(M3-FGF19^(C-tail)). The FGF1 portion includes mutations L⁴⁴F, M⁶⁷I,L⁷³V, V¹⁰⁹L, L¹¹¹I, C¹¹⁷V, A¹⁰³G, R¹¹⁹G, Δ¹⁰⁴⁻¹⁰⁶ and Δ¹²⁰⁻¹²². TheFGF19 portion is amino acids 119 to 166.

SEQ ID NO: 113 provides an exemplary FGF1 heparan binding KKK mutantanalog K112D, K113Q, K118V (wherein numbering refers to SEQ ID NO: 5).

SEQ ID NO: 114 provides an exemplary FGF1 heparan binding KKK mutantanalog with mutations K112D, K113Q, C117V, K118V (wherein numberingrefers to SEQ ID NO: 5).

SEQ ID NO: 115 provides an exemplary FGF1 heparan binding KKK mutantanalog with an N-terminal truncation and mutations K112D, K113Q, K118V(wherein numbering refers to SEQ ID NO: 5).

SEQ ID NO: 116 provides an exemplary FGF1 heparan binding KKK mutantanalog with an N-terminal truncation and mutations K112D, K113Q, K118V(wherein numbering refers to SEQ ID NO: 5).

SEQ ID NO: 117 provides an exemplary FGF1 heparan binding KKK mutantanalog with an N-terminal truncation and mutations K112D, K113Q, C117V,K118V (wherein numbering refers to SEQ ID NO: 5).

SEQ ID NO: 118 provides an exemplary FGF1 heparan binding KKK mutantanalog with an N-terminal truncation and mutations K112D, K113Q, C117V,K118V (wherein numbering refers to SEQ ID NO: 5).

SEQ ID NO: 119 provides an exemplary FGF1 heparan binding KKK mutantanalog with mutations K12V, N95V, K112D, K113Q, K118V (wherein numberingrefers to SEQ ID NO: 5).

SEQ ID NO: 120 provides an exemplary FGF1 heparan binding KKK mutantanalog with mutations K12V, N95V, K112D, K113Q, C117V, K118V (whereinnumbering refers to SEQ ID NO: 5).

SEQ ID NO: 121 provides an exemplary β-Klotho binding protein dimersequence (C2240) that can be attached at its N- or C-terminus directlyor indirectly to any of the FGF1 mutants provided herein to generate achimeric protein.

SEQ ID NO: 122 provides an exemplary β-Klotho binding protein sequencethat can be attached at its N- or C-terminus directly or indirectly toany of the FGF1 mutants provided herein to generate a chimeric protein.

SEQ ID NOs: 123-130 provide exemplary β-Klotho binding protein sequencesthat can be attached at their N- or C-termini directly or indirectly toany of the FGF1 mutants provided herein to generate a chimeric protein.In addition, each can be linked to SEQ ID NO: 122 via a linker and thenthe resulting chimera attached at its N- or C-terminus directly orindirectly to any of the FGF1 mutants provided herein to generate achimeric protein.

SEQ ID NOs: 131-140 provide exemplary β-Klotho binding protein sequencesthat can be attached at their N- or C-termini directly or indirectly toany of the FGF1 mutants provided herein to generate a chimeric protein.

SEQ ID NO: 141 provides an exemplary β-Klotho binding protein sequencethat can be attached at its N- or C-terminus directly or indirectly toany of the FGF1 mutants provided herein to generate a chimeric protein.In addition, it can be linked to any of SEQ ID NOS: 142-143 via a linkerand then the resulting chimera attached at its N- or C-terminus directlyor indirectly to any of the FGF1 mutants provided herein to generate achimeric protein.

SEQ ID NO: 142 provides an exemplary β-Klotho binding protein sequencethat can be attached at its N- or C-terminus directly or indirectly toany of the FGF1 mutants provided herein to generate a chimeric protein.In addition, it can be linked to SEQ ID NO: 141 via a linker and thenthe resulting chimera attached at its N- or C-terminus directly orindirectly to any of the FGF1 mutants provided herein to generate achimeric protein.

SEQ ID NO: 143 provides an exemplary β-Klotho binding protein sequencethat can be attached at its N- or C-terminus directly or indirectly toany of the FGF1 mutants provided herein to generate a chimeric protein.In addition, it can be linked to SEQ ID NO: 141 via a linker and thenthe resulting chimera attached at its N- or C-terminus directly orindirectly to any of the FGF1 mutants provided herein to generate achimeric protein.

SEQ ID NOs: 144-146 provide exemplary β-Klotho binding protein sequencesthat can be attached at their N- or C-termini directly or indirectly toany of the FGF1 mutants provided herein to generate a chimeric protein.

SEQ ID NO: 147 provides an exemplary FGFR1c binding protein sequencethat can be attached at its N- or C-terminus directly or indirectly toany of the FGF1 mutants provided herein to generate a chimeric protein.In addition, it can be linked to itself one or more times to generate anFGFR1c multimer, such as a dimer or a trimer.

SEQ ID NO: 148 (C2987) provides an exemplary FGFR1c binding proteinsequence that can be attached at its N- or C-terminus directly orindirectly to any of the FGF1 mutants provided herein to generate achimeric protein. In addition, it can be linked to itself one or moretimes to generate an FGFR1c multimer, such as a dimer or a trimer.

SEQ ID NOS: 149-167 provide exemplary FGFR1c binding protein sequencesthat can be attached at their N- or C-termini directly or indirectly toany of the FGF1 mutants provided herein to generate a chimeric protein.In addition, each can be linked to itself one or more times to generatean FGFR1c multimer, such as a dimer or a trimer, or combinations ofthese binding proteins can be linked together.

SEQ ID NOs: 168-171 provide exemplary β-Klotho-FGFR1c binding proteinsequences that can be attached at their N- or C-termini directly orindirectly to any of the FGF1 mutants provided herein to generate achimeric protein.

SEQ ID NO: 172 provides an exemplary WT-FGF1/β-Klotho binding proteinchimera sequence (C2240). This is represented in FIG. 25A.

SEQ ID NO: 173 provides an exemplary ΔNT FGF1/β-Klotho binding proteinchimera sequence (C2240). This is represented in FIG. 25B.

SEQ ID NO: 174 provides an exemplary FGF1 KN/β-Klotho binding proteinchimera sequence (C2240). This is represented in FIG. 25C.

SEQ ID NO: 175 provides an exemplary FGF1KKK/β-Klotho binding proteinchimera sequence (C2240). This is represented in FIG. 25D.

SEQ ID NO: 176 provides an exemplary WT-FGF1/β-Klotho binding proteinchimera sequence (C2240) with two β-Klotho binding protein portions.This is represented in FIG. 25F.

SEQ ID NO: 177 provides an exemplary ΔNT FGF1/β-Klotho binding proteinchimera sequence (C2240) with two β-Klotho binding protein portions.This is represented in FIG. 25G.

SEQ ID NO: 178 provides an exemplary FGF1 KN/β-Klotho binding proteinchimera sequence (C2240) with two β-Klotho binding protein portions.This is represented in FIG. 25H.

SEQ ID NO: 179 provides an exemplary FGF1 KKK/β-Klotho binding proteinchimera sequence (C2240) with two β-Klotho binding protein portions.This is represented in FIG. 25I.

SEQ ID NO: 180 provides an exemplary WT-FGF1/β-Klotho binding proteinchimera sequence (C2240). This is represented in FIG. 26A.

SEQ ID NO: 181 provides an exemplary ΔNT FGF1/β-Klotho binding proteinchimera sequence (C2240). This is represented in FIG. 26B.

SEQ ID NO: 182 provides an exemplary FGF1 KN/β-Klotho binding proteinchimera sequence (C2240). This is represented in FIG. 26C.

SEQ ID NO: 183 provides an exemplary FGF1KKK/β-Klotho binding proteinchimera sequence (C2240). This is represented in FIG. 26D.

SEQ ID NO: 184 provides an exemplary WT-FGF1/β-Klotho binding proteinchimera sequence (C2240) with two β-Klotho binding protein portions.This is represented in FIG. 26F.

SEQ ID NO: 185 provides an exemplary dNT FGF1/β-Klotho binding proteinchimera sequence (C2240) with two β-Klotho binding protein portions.This is represented in FIG. 26F.

SEQ ID NO: 186 provides an exemplary FGF1 KN/β-Klotho binding proteinchimera sequence (C2240) with two β-Klotho binding protein portions.This is represented in FIG. 25H.

SEQ ID NO: 187 provides an exemplary FGF1KKK/β-Klotho binding proteinchimera sequence (C2240) with two β-Klotho binding protein portions.This is represented in FIG. 25I.

SEQ ID NO: 188 provides an exemplary ΔNT FGF1/FGFR1c-binding proteinchimera sequence (C2987). This is represented in FIG. 23J.

SEQ ID NO: 189 provides an exemplary ΔNT FGF1/FGFR1c-binding proteinchimera sequence (C2987). This is represented in FIG. 24I.

SEQ ID NO: 190 provides an exemplary FGFR1c dimer chimera sequence(C2987). This is represented in FIG. 25E.

SEQ ID NO: 191 (FGF1(1-140αα) R35E, C117V) provides an exemplary matureform of FGF1 with mutations (R35E and C117V, wherein numbering refers toSEQ ID NO: 5) to reduce mitogenic activity and increase thermostability.

SEQ ID NO: 192 (FGF1(1-140αα) R35E, C117V, KKK) provides an exemplarymature form of FGF1 with mutations (R35E, K112D, K113Q, C117V, and K118Vwherein numbering refers to SEQ ID NO: 5) to reduce mitogenic activityand increase thermostability.

SEQ ID NO: 193 (FGF1(1-140αα) R35E, C117V K12V, N95V) provides anexemplary mature form of FGF1 with mutations (K12V, R35E, N95V, andC117V wherein numbering refers to SEQ ID NO: 5) to reduce mitogenicactivity and increase thermostability.

SEQ ID NO: 194 (FGF1^(ΔNT1) (10-140αα) R35E, C117V) provides anexemplary N-terminally truncated form of FGF1 with mutations (R35E andC117V, wherein numbering refers to SEQ ID NO: 5) to reduce mitogenicactivity and increase thermostability.

SEQ ID NO: 195 (FGF1^(ΔNTKN) KKK (10-140αα)) provides an exemplaryN-terminally truncated form of FGF1 with mutations (K112D, K113Q, K118V,K12V, N95V, C117V, and R35E, wherein numbering refers to SEQ ID NO: 5)to reduce mitogenic activity and increase thermostability.

SEQ ID NO: 196 (FGF1 KKK (KN) (1-140αα)) provides an exemplary matureform of FGF1 with mutations (K112D, K113Q, K118V, K12V, N95V, C117V, andR35E, wherein numbering refers to SEQ ID NO: 5) to reduce mitogenicactivity and increase thermostability.

SEQ ID NO: 197 (FGF1^(ΔNT1) (10-140αα) M2KN) provides an exemplaryN-terminally truncated form of FGF1 with mutations (K12V, L44F, R35E,C83T, N95V, C117V, and F132W, wherein numbering refers to SEQ ID NO: 5)to reduce mitogenic activity and increase thermostability.

SEQ ID NO: 198 (FGF1^(ΔNT1) (10-140αα) M2KNKKK) provides an exemplaryN-terminally truncated form of FGF1 with mutations (K12V, L44F, R35E,C83T, N95V, C117V, K112D, K113Q, K118V, and F132W, wherein numberingrefers to SEQ ID NO: 5) to reduce mitogenic activity and increasethermostability.

SEQ ID NO: 199 (FGF1(1-140αα) R35V, C117V) provides an exemplary matureform of FGF1 with mutations (R35V and C117V, wherein numbering refers toSEQ ID NO: 5) to reduce mitogenic activity and increase thermostability.

SEQ ID NO: 200 (FGF1(1-140αα) R35V, C117V, KKK) provides an exemplarymature form of FGF1 with mutations (R35V, K112D, K113Q, C117V, and K118Vwherein numbering refers to SEQ ID NO: 5) to reduce mitogenic activityand increase thermostability.

SEQ ID NO: 201 (FGF1(1-140αα) K12V, R35V, N95V, C117V) provides anexemplary mature form of FGF1 with mutations (K12V, R35V, N95V, andC117V wherein numbering refers to SEQ ID NO: 5) to reduce mitogenicactivity and increase thermostability.

SEQ ID NO: 202 (FGF1^(ΔNT1) (10-140αα) R35V, C117V) provides anexemplary N-terminally truncated form of FGF1 with mutations (R35V andC117V, wherein numbering refers to SEQ ID NO: 5) to reduce mitogenicactivity and increase thermostability.

SEQ ID NO: 203 (FGF1^(ΔNTKNT) KKK (10-140αα)) provides an exemplaryN-terminally truncated form of FGF1 with mutations (K112D, K113Q, K118VK12V, N95V, C117V, and R35V, wherein numbering refers to SEQ ID NO: 5)to reduce mitogenic activity and increase thermostability.

SEQ ID NO: 204 (FGF1 KKK (KN) (1-140αα)) provides an exemplary matureform of FGF1 with mutations (K112D, K113Q, K118V, K12V, N95V, C117V, andR35V, wherein numbering refers to SEQ ID NO: 5) to reduce mitogenicactivity and increase thermostability.

SEQ ID NO: 205 (FGF1^(ΔNT1) (10-140αα)M2KN) provides an exemplaryN-terminally truncated form of FGF1 with mutations (K12V, L44F, R35V,C83T, N95V, C117V, and F132W, wherein numbering refers to SEQ ID NO: 5)to reduce mitogenic activity and increase thermostability.

SEQ ID NO: 206 (FGF1^(ΔNT1) (10-140αα) M2KNKKK) provides an exemplaryN-terminally truncated form of FGF1 with mutations (K12V, L44F, R35V,C83T, N95V, C117V, K112D, K113Q, K118V, and F132W, wherein numberingrefers to SEQ ID NO: 5) to reduce mitogenic activity and increasethermostability.

SEQ ID NO: 207 (FGF1-140αα) C117V, KKKR provides an exemplary matureform of FGF1 with mutations (K112D, K113Q, C117V, K118V, R119V, whereinnumbering refers to SEQ ID NO: 5) to reduce mitogenic activity andincrease thermostability.

SEQ ID NO: 208 (FGF1-140αα) C117V, KY provides an exemplary mature formof FGF1 with mutations (K12V, Y94V, C117V, wherein numbering refers toSEQ ID NO: 5) to reduce mitogenic activity and increase thermostability.

SEQ ID NO: 209 (FGF1-140αα) C117V, KE provides an exemplary mature formof FGF1 with mutations (K12V, E87V, C117V, wherein numbering refers toSEQ ID NO: 5) to reduce mitogenic activity and increase thermostability.

SEQ ID NO: 210 (FGF1-140αα) C117V, KEY provides an exemplary mature formof FGF1 with mutations (K12V, E87V, Y94V, C117V, wherein numberingrefers to SEQ ID NO: 5) to reduce mitogenic activity and increasethermostability.

SEQ ID NO: 211 (FGF1-140αα) C117V, KNY provides an exemplary mature formof FGF1 with mutations (K12V, Y94V, N95V, C117V, wherein numberingrefers to SEQ ID NO: 5) to reduce mitogenic activity and increasethermostability.

SEQ ID NO: 212 (FGF1-140αα) K12V, L46V, E87V, N95V, C117V, P134Vprovides an exemplary mature form of FGF1 with point mutations (K12V,L46V, E87V, N95V, C117V, P134V, wherein numbering refers to SEQ ID NO:5) to reduce mitogenic activity and increase thermostability.

SEQ ID NO: 213 (FGF1-140αα) C117V, K118V provides an exemplary matureform of FGF1 with mutations (C117V and K118V, wherein numbering refersto SEQ ID NO: 5) to reduce mitogenic activity and increasethermostability.

SEQ ID NO: 214 (FGF^(ΔNT1C) 10-140αα) K12V, N95V, C83T, C117V providesan exemplary N-terminally truncated form of FGF1 with mutations (K12V,N95V, C83T, and C117V, wherein numbering refers to SEQ ID NO: 5) toreduce mitogenic activity and increase thermostability.

SEQ ID NO: 215 (FGF^(ΔNT1C) 10-140αα) K12V, N95V, C16T, C83S, C117A,provides an exemplary N-terminally truncated form of FGF1 with mutations(K12V, N95V, C16T, C83S, and C117A, wherein numbering refers to SEQ IDNO: 5) to reduce mitogenic activity and increase thermostability.

SEQ ID NO: 216 (FGF^(ΔNT1) 10-140αα) H21Y, L44F, H102Y, F108Y, C117V,provides an exemplary N-terminally truncated form of FGF1 with mutations(H21Y, L44F, H102Y, F108Y, and C117V, wherein numbering refers to SEQ IDNO: 5) to reduce mitogenic activity and increase thermostability.

SEQ ID NO: 217 (FGF^(ΔNT1) 10-140αα) K12V, H21Y, L44F, N95V, H102Y,F108Y, C117V, provides an exemplary N-terminally truncated form of FGF1with mutations (K12V, H21Y, L44F, N95V, H102Y, F108Y, and C117V, whereinnumbering refers to SEQ ID NO: 5) to reduce mitogenic activity andincrease thermostability.

SEQ ID NO: 218 (FGF1 1-140αα) K12V, H21Y, L44F, N95V, H102Y, F108Y,C117V, provides an exemplary mature form of FGF1 with mutations (K12V,H21Y, L44F, N95V, H102Y, F108Y, and C117V, wherein numbering refers toSEQ ID NO: 5) to reduce mitogenic activity and increase thermostability.

SEQ ID NO: 219 (wtFGF1ΔHBS-FGF21C-tail) provides an exemplary matureform of FGF1 with mutations that reduce the functionality of the heparinbinding site to affect serum half-life and receptor affinity (K112D,K113Q, K118V, wherein numbering refers to SEQ ID NO: 5) fused to aportion of FGF21 at the C-terminus (amino acids 136 to 177) to generatea reagent that combines the metabolic benefits of a β klotho-dependentagonist (FGF21) and β klotho-independent agonist (FGF1).

SEQ ID NO: 220 (wtFGF1ΔHBS-FGF19C-tail) provides an exemplary matureform of FGF1 with mutations that reduce the functionality of the heparinbinding site to affect serum half-life and receptor affinity (K112D,K113Q, K118V, wherein numbering refers to SEQ ID NO: 5) fused to aportion of FGF19 at the C-terminus (amino acids 138 to 183) to generatea reagent that combines the metabolic benefits of a β klotho-dependentagonist (FGF19) and β klotho-independent agonist (FGF1).

SEQ ID NO: 221 provides an exemplary N-terminally truncated form ofFGF1, wherein the 16 N-terminal amino acids are from FGF21 (amino acids28-43 of SEQ ID NO: 20), and the sequence includes a C117V mutation.

SEQ ID NO: 222 provides an exemplary N-terminally truncated form ofFGF1, wherein the four N-terminal amino acids are from FGF21 (aminoacids 40-43 of SEQ ID NO: 20), and the sequence includes a C117Vmutation.

SEQ ID NO: 223 (wtFGF1-FGF21C-tail) provides an exemplary mature form ofFGF1 fused to a portion of FGF21 at the C-terminus (amino acids 136 to177) to generate a reagent that combines the metabolic benefits of a βklotho-dependent agonist (FGF21) and β klotho-independent agonist(FGF1).

SEQ ID NO: 224 (wtFGF1-FGF19C-tail) provides an exemplary mature form ofFGF1 fused to a portion of FGF19 at the C-terminus (amino acids 138 to183) to generate a reagent that combines the metabolic benefits of a βklotho-dependent agonist (FGF19) and β klotho-independent agonist(FGF1).

SEQ ID NO: 225 (FGF^(ΔNT1C) 10-140αα) K12V, N95V, C117V, provides anexemplary N-terminally truncated form of FGF1 with mutations (K12V,N95V, and C117V, wherein numbering refers to SEQ ID NO: 5) to reduce themitogenicity and increase the stability of FGF1.

SEQ ID NO: 226 (FGF1 KKK 1-140αα) K112D, K113Q, K118V, provides anexemplary mature form of FGF1 with mutations (K112D, K113Q, and K118V,wherein numbering refers to SEQ ID NO: 5) to reduce the mitogenicity andincrease the stability of FGF1.

SEQ ID NO: 227 (FGF1 1-140αα) K12V, Q40P, S47I, H93G, N95V, provides anexemplary mature form of FGF1 with mutations (K12V, Q40P, S47I, H93G,and N95V, wherein numbering refers to SEQ ID NO: 5) to reduce themitogenicity and increase the thermal stability of FGF1.

SEQ ID NO: 228 (FGF^(ΔNT) 10-140αα) K12V, Q40P, S47I, H93G, N95Vprovides an exemplary N-terminally truncated form of FGF1 with mutations(K12V, Q40P, S47I, H93G, and N95V, wherein numbering refers to SEQ IDNO: 5) to reduce the mitogenicity and increase the thermal stability ofFGF1.

SEQ ID NO: 229 (FGF1 1-140αα) M2KN K12V, L44F, C83T, N95V, C117V, F132Wprovides an exemplary mature form of FGF1 with mutations (K12V, L44F,C83T, N95V, C117V, and F132W, wherein numbering refers to SEQ ID NO: 5)to reduce the mitogenicity without increasing the thermal stability ofFGF1.

SEQ ID NO: 230 (FGF1 1-140αα) C117V provides an exemplary mature form ofFGF1 with mutation (C117V, wherein numbering refers to SEQ ID NO: 5) toimprove the stability of FGF1 by eliminating a free cysteine the canform disulfide brigded aggregated protein.

SEQ ID NO: 231 (FGF1 1-140αα))KKK(KN) K112D, K113Q, K118V, K12V, N95V,C117V provides an exemplary mature form of FGF1 with mutations (K112D,K113Q, K118V, K12V, N95V, and C117V, wherein numbering refers to SEQ IDNO: 5) to reduce mitogenicity and heparan binding, and decrease thepotential for protein aggregation of FGF1.

SEQ ID NO: 232 (FGF1 10-140αα) M2KN K12V, L44F, C83T, N95V, C117V,F132W, provides an exemplary N-terminally truncated form of FGF1 withmutations (K12V, L44F, C83T, N95V, C117V, and F132W, wherein numberingrefers to SEQ ID NO: 5) to reduce mitogenicity and decrease thepotential for protein aggregation of FGF1, without affecting the thermalstability.

SEQ ID NO: 233 (FGF1 1-140αα) R35E, C117V, provides an exemplary matureform of FGF1 with mutations (R35E and C117V, wherein numbering refers toSEQ ID NO: 5) to manipulate the receptor binding affinity/specificityand decrease the potential for protein aggregation of FGF1.

SEQ ID NO: 234 (FGF1 1-140αα) KY K12V, Y94V, C117V, provides anexemplary mature form of FGF1 with mutations (K12V, Y94V, and C117V,wherein numbering refers to SEQ ID NO: 5) to manipulate the receptorbinding affinity/specificity and decrease the potential for proteinaggregation of FGF1.

SEQ ID NO: 235 (FGF1 1-140αα) KE K12V, E87V, C117V, provides anexemplary mature form of FGF1 with mutations (K12V, E87V, and C117V,wherein numbering refers to SEQ ID NO: 5) to manipulate the receptorbinding affinity/specificity and decrease the potential for proteinaggregation of FGF1

SEQ ID NO: 236 (FGF1 1-140αα) KKKR K112D, K113Q, C117V, K118V, R119Vprovides an exemplary mature form of FGF1 with mutations (K112D, K113Q,C117V, K118V, and R119V, wherein numbering refers to SEQ ID NO: 5) toreduce the heparan binding affinity/specificity and decrease thepotential for protein aggregation of FGF1.

SEQ ID NO: 237 (FGF1 1-140αα) KN R35E, K12V, N95V, C117V provides anexemplary mature form of FGF1 with mutations (R35E, K12V, N95V, andC117V, wherein numbering refers to SEQ ID NO: 5) to manipulate thereceptor binding affinity/specificity and decrease the potential forprotein aggregation of FGF1.

SEQ ID NO: 238 (FGF1 10-140αα) KN R35E, C117V provides an exemplaryN-terminally truncated form of FGF1 with mutations (R35E and C117Vwherein numbering refers to SEQ ID NO: 5) to manipulate the receptorbinding affinity/specificity and decrease the potential for proteinaggregation of FGF1.

DETAILED DESCRIPTION

The following explanations of terms and methods are provided to betterdescribe the present disclosure and to guide those of ordinary skill inthe art in the practice of the present disclosure. The singular forms“a,” “an,” and “the” refer to one or more than one, unless the contextclearly dictates otherwise. For example, the term “comprising a cell”includes single or plural cells and is considered equivalent to thephrase “comprising at least one cell.” The term “or” refers to a singleelement of stated alternative elements or a combination of two or moreelements, unless the context clearly indicates otherwise. As usedherein, “comprises” means “includes.” Thus, “comprising A or B,” means“including A, B, or A and B,” without excluding additional elements.Dates of GenBank® Accession Nos. referred to herein are the sequencesavailable at least as early as Oct. 21, 2013. All references andGenBank® Accession numbers cited herein are incorporated by reference.

Unless explained otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood to one of ordinaryskill in the art to which this disclosure belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described below. The materials, methods, and examples areillustrative only and not intended to be limiting.

In order to facilitate review of the various embodiments of thedisclosure, the following explanations of specific terms are provided:

Administration: To provide or give a subject an agent, such as a mutatedFGF1 protein disclosed herein, by any effective route. Exemplary routesof administration include, but are not limited to, oral, injection (suchas subcutaneous, intramuscular, intradermal, intraperitoneal,intravenous, and intratumoral), sublingual, rectal, transdermal,intranasal, vaginal and inhalation routes.

Beta-Klotho binding domain or protein: A peptide sequence that bindsselectively to β-Klotho (such as human β-Klotho, OMIM 61135, GenBank®Accession No. NP_(—)783864.1), but not to other proteins. β-Klotho is acofactor for FGF21 activity. Such a binding domain can include one ormore monomers (wherein the monomers can be the same or differentβ-Klotho binding proteins), thereby generating a multimer (such as adimer). In specific examples, such a domain/protein is not an antibody.Exemplary β-Klotho binding proteins can be found in SEQ ID NOS: 121,122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135,136, 137, 138, 139, 140, 141, 142, 143, 144, 145 146, and 168-171 aswell as U.S. Pat. No. 8,372,952, U.S. Publication No. 2013/0197191, andSmith et al., PLoS One 8:e61432, 2013, all herein incorporated byreference.

A β-Klotho binding protein “specifically binds” to β-Klotho when thedissociation constant (K_(D)) is at least about 1×10⁻⁷ M, at least about1.5×10⁻⁷, at least about 2×10⁻⁷, at least about 2.5×10⁻⁷, at least about3×10⁻⁷, at least about at least about 5×10⁻⁷ M, at least about 1×10⁻⁸ M,at least about 5×10⁻⁸, at least about 1×10⁻⁹, at least about 5×10⁻⁹, atleast about 1×10⁻¹⁰, or at least about 5×10⁻¹⁰ M. In one embodiment,K_(D) is measured by a radiolabeled antigen binding assay (RIA)performed with the β-Klotho binding protein and β-Klotho. In anotherexample, K_(D) is measured using an ELISA assay.

C-terminal portion: A region of a protein sequence that includes acontiguous stretch of amino acids that begins at or near the C-terminalresidue of the protein. A C-terminal portion of the protein can bedefined by a contiguous stretch of amino acids (e.g., a number of aminoacid residues).

Chimeric protein: A protein that includes at least a portion of thesequence of a full-length first protein (e.g., FGF1) and at least aportion of the sequence of a full-length second protein (e.g., FGF19,FGF21, β-Klotho-binding protein, or FGF1Rc-binding protein), where thefirst and second proteins are different. A chimeric polypeptide alsoencompasses polypeptides that include two or more non-contiguousportions derived from the same polypeptide. The two different peptidescan be joined directly or indirectly, for example using a linker.

Diabetes mellitus: A group of metabolic diseases in which a subject hashigh blood sugar, either because the pancreas does not produce enoughinsulin, or because cells do not respond to the insulin that isproduced. Type 1 diabetes results from the body's failure to produceinsulin. This form has also been called “insulin-dependent diabetesmellitus” (IDDM) or “juvenile diabetes”. Type 2 diabetes results frominsulin resistance, a condition in which cells fail to use insulinproperly, sometimes combined with an absolute insulin deficiency. Thisform is also called “non insulin-dependent diabetes mellitus” (NIDDM) or“adult-onset diabetes.” The defective responsiveness of body tissues toinsulin is believed to involve the insulin receptor. Diabetes mellitusis characterized by recurrent or persistent hyperglycemia, and in someexamples diagnosed by demonstrating any one of:

-   -   a. Fasting plasma glucose level≧7.0 mmol/l (126 mg/dl);    -   b. Plasma glucose≧11.1 mmol/l (200 mg/dL) two hours after a 75 g        oral glucose load as in a glucose tolerance test;    -   c. Symptoms of hyperglycemia and casual plasma glucose≧11.1        mmol/l (200 mg/dl);    -   d. Glycated hemoglobin (Hb A1C)≧6.5%

Effective amount or Therapeutically effective amount: The amount ofagent, such as a mutated FGF1 protein (or nucleic acid encoding such)disclosed herein, that is an amount sufficient to prevent, treat(including prophylaxis), reduce and/or ameliorate the symptoms and/orunderlying causes of any of a disorder or disease. In one embodiment, an“effective amount” is sufficient to reduce or eliminate a symptom of adisease, such as a diabetes (such as type II diabetes), for example bylowering blood glucose.

Fibroblast Growth Factor 1 (FGF1): OMIM 13220. Includes FGF1 nucleicacid molecules and proteins. A protein that binds to the FGF receptor,and is also known as the acidic FGF. FGF1 sequences are publicallyavailable, for example from GenBank® sequence database (e.g., AccessionNos. NP_(—)00791 and NP_(—)034327 provide exemplary FGF1 proteinsequences, while Accession Nos. NM_(—)000800 and NM_(—)010197 provideexemplary FGF1 nucleic acid sequences). One of ordinary skill in the artcan identify additional FGF1 nucleic acid and protein sequences,including FGF1 variants.

Specific examples of native FGF1 sequences are provided in SEQ ID NOS:1-5. A native FGF1 sequence is one that does not include a mutation thatalters the normal activity of the protein (e.g., activity of SEQ ID NO:2, 4 or SEQ ID NO: 5). A mutated FGF1 is a variant of FGF1 withdifferent or altered biological activity, such as reduced mitogenicity(e.g., a variant of any of SEQ ID NOS: 1-5, such as one having at least90%, at least 95%, at least 96%, at least 97%, at least 98% or at least99% sequence identity to any of SEQ ID NOS: 1-5, but is not anative/wild-type sequence). In one example, such a variant includes anN-terminal truncation, at least one point mutation (such as one or moreof those shown in Table 1), or combinations thereof, such as changesthat decrease mitogenicity of FGF1. Mutated FGF1 proteins include FGF1chimeras (e.g., FGF1/FGF19 chimeras). Specific exemplary FGF1 mutantproteins are shown in SEQ ID NOS: 6-13, 6, 7, 8, 9, 10, 11, 12, 13, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 113, 114, 115, 116, 117, 118, 119,120, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203,204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217,218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231,232, 233, 234, 235, 236, 237 and 238.

Fibroblast Growth Factor 19 (FGF19): OMIM 603891. Includes FGF19 nucleicacid molecules and proteins. FGF19 regulates bile acid synthesis and haseffects on glucose and lipid metabolism. FGF19 sequences are publicallyavailable, for example from the GenBank® sequence database (e.g.,Accession Nos. NP_(—)005108.1 and AAQ88669.1 provide exemplary FGF19protein sequences, while Accession Nos. AY358302.1 and NM_(—)005117.2provide exemplary FGF19 nucleic acid sequences). One of ordinary skillin the art can identify additional FGF19 nucleic acid and proteinsequences, including FGF19 variants.

Fibroblast Growth Factor 21 (FGF21): OMIM 609436. Includes FGF21 nucleicacid molecules and proteins. FGF21 stimulates glucose updated inadipocytes. FGF21 sequences are publically available, for example fromthe GenBank® sequence database (e.g., Accession Nos. AAQ89444.1,NP_(—)061986, and AAH49592.1 provide exemplary FGF21 protein sequences,while Accession Nos. AY359086.1 and BC049592 provide exemplary FGF21nucleic acid sequences). One of ordinary skill in the art can identifyadditional FGF21 nucleic acid and protein sequences, including FGF21variants.

Fibroblast Growth Factor Receptor 1c (FGFR1c) binding domain or protein:A peptide sequence that binds selectively to FGFR1c (such as humanFGFR1c, e.g., GeneBank Accession No. NP_(—)001167536.1 orNP_(—)056934.2), but not to other proteins. FGFR1c is a cofactor forFGF21 activity. Such a binding domain can include one or more monomers(wherein the monomers can be the same or different sequences), therebygenerating a multimer (such as a dimer). In specific examples, such adomain/protein is not an antibody. Exemplary FGFR1c-binding proteins canbe found in SEQ ID NOS: 147, 148, 149, 150, 151, 152, 153, 154, 155,156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167 and portionsof 168, 169, 170 and 171, or a multimer thereof such as SEQ ID NO: 190,as well as U.S. Pat. No. 8,372,952, U.S. Publication No. 2013/0197191,and Smith et al., PLoS One 8:e61432, 2013, all herein incorporated byreference. Thus, reference to a FGFR1c-binding protein multimer,includes proteins made using two or more peptides having at least 90%,at least 95%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100% sequence identity to one or more of SEQ ID NO: 147,148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161,162, 163, 164, 165, 166, 167, and 190.

A FGFR1c binding protein “specifically binds” to FGFR1c when thedissociation constant (KD) is at least about 1×10⁻⁷ M, at least about1.5×10⁻⁷, at least about 2×10⁻⁷, at least about 2.5×10⁻⁷, at least about3×10⁻⁷, at least about at least about 5×10⁻⁷ M, at least about 1×10⁻⁸ M,at least about 5×10⁻⁸, at least about 1×10⁻⁹, at least about 5×10⁻⁹, atleast about 1×10⁻¹⁰, or at least about 5×10⁻¹⁰ M. In one embodiment, KDis measured by a radiolabeled antigen binding assay (RIA) performed withthe FGFR1c-binding protein and FGFR1c. In another example, K_(D) ismeasured using an ELISA assay.

Fibroblast Growth Factor Receptor 1c (FGFR1c): Also known as FGFR1isoform 2. Includes FGFR1c nucleic acid molecules and proteins. FGFR1cand β-Klotho can associate with FGF21 to form a signaling complex.FGFR1c sequences are publically available, for example from the GenBank®sequence database (e.g., Accession Nos. NP_(—)001167536.1 andNP_(—)056934.2 provide exemplary FGFR1c protein sequences). One ofordinary skill in the art can identify additional FGFR1c nucleic acidand protein sequences, including FGFR1c variants.

Fibroblast Growth Factor Receptor 4 (FGFR4): OMIM 134935. Includes FGFR4nucleic acid molecules and proteins. FGFR4 can bind to some FGFproteins, including FGF1. FGFR4 sequences are publically available, forexample from the GenBank® sequence database (e.g., Accession Nos.NM_(—)002011 and AAB25788.1 provide exemplary FGFR4 protein sequences,while Accession Nos. NM_(—)002002 and L03840.1 provide exemplary FGFR4nucleic acid sequences). One of ordinary skill in the art can identifyadditional FGFR4 nucleic acid and protein sequences, including FGFR4variants.

Host cells: Cells in which a vector can be propagated and its DNAexpressed. The cell may be prokaryotic or eukaryotic. The term alsoincludes any progeny of the subject host cell. It is understood that allprogeny may not be identical to the parental cell since there may bemutations that occur during replication. However, such progeny areincluded when the term “host cell” is used. Thus, host cells can betransgenic, in that they include nucleic acid molecules that have beenintroduced into the cell, such as a nucleic acid molecule encoding amutant FGF1 protein disclosed herein.

Isolated: An “isolated” biological component (such as a mutated FGF1protein or nucleic acid molecule) has been substantially separated,produced apart from, or purified away from other biological componentsin the cell of the organism in which the component naturally occurs,such as other chromosomal and extrachromosomal DNA and RNA, andproteins. Nucleic acids molecules and proteins which have been“isolated” thus include nucleic acids and proteins purified by standardpurification methods. The term also embraces nucleic acid molecules andproteins prepared by recombinant expression in a host cell as well aschemically synthesized nucleic acids. A purified or isolated cell,protein, or nucleic acid molecule can be at least 70%, at least 80%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% pure.

Linker. A moiety or group of moieties that joins or connects two or morediscrete separate peptide or proteins, such as monomer domains, forexample to generate a chimeric protein. In one example a linker is asubstantially linear moiety. Exemplary linkers that can be used togenerate the chimeric proteins provided herein include but are notlimited to: peptides, nucleic acid molecules, peptide nucleic acids, andoptionally substituted alkylene moieties that have one or more oxygenatoms incorporated in the carbon backbone. A linker can be a portion ofa native sequence, a variant thereof, or a synthetic sequence. Linkerscan include naturally occurring amino acids, non-naturally occurringamino acids, or a combination of both. In one example a linker iscomposed of at least 5, at least 10, at least 15 or at least 20 aminoacids, such as 5 to 10, 5 to 20, or 5 to 50 amino acids. In one examplethe linker is a poly alanine.

Mammal: This term includes both human and non-human mammals. Similarly,the term “subject” includes both human and veterinary subjects (such ascats, dogs, cows, and pigs) and rodents (such as mice and rats).

Metabolic disorder/disease: A disease or disorder that results from thedisruption of the normal mammalian process of metabolism. Includesmetabolic syndrome.

Examples include but are not limited to: (1) glucose utilizationdisorders and the sequelae associated therewith, including diabetesmellitus (Type I and Type-2), gestational diabetes, hyperglycemia,insulin resistance, abnormal glucose metabolism, “pre-diabetes”(Impaired Fasting Glucose (IFG) or Impaired Glucose Tolerance (IGT)),and other physiological disorders associated with, or that result from,the hyperglycemic condition, including, for example, histopathologicalchanges such as pancreatic β-cell destruction; (2) dyslipidemias andtheir sequelae such as, for example, atherosclerosis, coronary arterydisease, cerebrovascular disorders and the like; (3) other conditionswhich may be associated with the metabolic syndrome, such as obesity andelevated body mass (including the co-morbid conditions thereof such as,but not limited to, nonalcoholic fatty liver disease (NAFLD),nonalcoholic steatohepatitis (NASH), and polycystic ovarian syndrome(PCOS)), and also include thromboses, hypercoagulable and prothromboticstates (arterial and venous), hypertension, cardiovascular disease,stroke and heart failure; (4) disorders or conditions in whichinflammatory reactions are involved, including atherosclerosis, chronicinflammatory bowel diseases (e.g., Crohn's disease and ulcerativecolitis), asthma, lupus erythematosus, arthritis, or other inflammatoryrheumatic disorders; (5) disorders of cell cycle or cell differentiationprocesses such as adipose cell tumors, lipomatous carcinomas including,for example, liposarcomas, solid tumors, and neoplasms; (6)neurodegenerative diseases and/or demyelinating disorders of the centraland peripheral nervous systems and/or neurological diseases involvingneuroinfiammatory processes and/or other peripheral neuropathies,including Alzheimer's disease, multiple sclerosis, Parkinson's disease,progressive multifocal leukoencephalopathy and Guillian-Barre syndrome;(7) skin and dermatological disorders and/or disorders of wound healingprocesses, including erythemato-squamous dermatoses; and (8) otherdisorders such as syndrome X, osteoarthritis, and acute respiratorydistress syndrome. Other examples are provided in WO 2014/085365 (hereinincorporated by reference).

In specific examples, the metabolic disease includes one or more of(such as at least 2 or at least 3 of): diabetes (such as type 2diabetes, non-type 2 diabetes, type 1 diabetes, latent autoimmunediabetes (LAD), or maturity onset diabetes of the young (MODY)),polycystic ovary syndrome (PCOS), metabolic syndrome (MetS), obesity,non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease(NAFLD), dyslipidemia (e.g., hyperlipidemia), and cardiovasculardiseases (e.g., hypertension).

N-terminal portion: A region of a protein sequence that includes acontiguous stretch of amino acids that begins at or near the N-terminalresidue of the protein. An N-terminal portion of the protein can bedefined by a contiguous stretch of amino acids (e.g., a number of aminoacid residues).

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence (such as a mutated FGF1 coding sequence). Generally,operably linked DNA sequences are contiguous and, where necessary tojoin two protein coding regions, in the same reading frame.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers useful in this invention are conventional. Remington'sPharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton,Pa., 15th Edition (1975), describes compositions and formulationssuitable for pharmaceutical delivery of the disclosed mutated FGF1proteins and FGFR1c-binding protein multimers (or nucleic acid moleculesencoding such) herein disclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

Promoter: An array of nucleic acid control sequences which directtranscription of a nucleic acid. A promoter includes necessary nucleicacid sequences near the start site of transcription, such as, in thecase of a polymerase II type promoter, a TATA element. A promoter alsooptionally includes distal enhancer or repressor elements which can belocated as much as several thousand base pairs from the start site oftranscription.

Recombinant: A recombinant nucleic acid molecule is one that has asequence that is not naturally occurring (e.g., a mutated FGF1 orchimeric protein) or has a sequence that is made by an artificialcombination of two otherwise separated segments of sequence. Thisartificial combination can be accomplished by routine methods, such aschemical synthesis or by the artificial manipulation of isolatedsegments of nucleic acids, such as by genetic engineering techniques.Similarly, a recombinant protein is one encoded for by a recombinantnucleic acid molecule. Similarly, a recombinant or transgenic cell isone that contains a recombinant nucleic acid molecule and expresses arecombinant protein.

Sequence identity of amino acid sequences: The similarity between aminoacid (or nucleotide) sequences is expressed in terms of the similaritybetween the sequences, otherwise referred to as sequence identity.Sequence identity is frequently measured in terms of percentage identity(or similarity or homology); the higher the percentage, the more similarthe two sequences are. Homologs or variants of a polypeptide willpossess a relatively high degree of sequence identity when aligned usingstandard methods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J.Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci.U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins andSharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994, presents adetailed consideration of sequence alignment methods and homologycalculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403, 1990) is available from several sources, includingthe National Center for Biotechnology Information (NCBI, Bethesda, Md.)and on the internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. A description ofhow to determine sequence identity using this program is available onthe NCBI website on the internet.

Homologs and variants of the mutated FGF1 proteins and coding sequencesdisclosed herein are typically characterized by possession of at leastabout 80%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98% or at least 99% sequence identity counted over the full lengthalignment with the amino acid sequence using the NCBI Blast 2.0, gappedblastp set to default parameters. For comparisons of amino acidsequences of greater than about 30 amino acids, the Blast 2 sequencesfunction is employed using the default BLOSUM62 matrix set to defaultparameters, (gap existence cost of 11, and a per residue gap cost of 1).When aligning short peptides (fewer than around 30 amino acids), thealignment should be performed using the Blast 2 sequences function,employing the PAM30 matrix set to default parameters (open gap 9,extension gap 1 penalties). Proteins with even greater similarity to thereference sequences will show increasing percentage identities whenassessed by this method, such as at least 95%, at least 98%, or at least99% sequence identity. When less than the entire sequence is beingcompared for sequence identity, homologs and variants will typicallypossess at least 80% sequence identity over short windows of 10-20 aminoacids, and may possess sequence identities of at least 85% or at least90% or at least 95% depending on their similarity to the referencesequence. Methods for determining sequence identity over such shortwindows are available at the NCBI website on the internet. One of skillin the art will appreciate that these sequence identity ranges areprovided for guidance only; it is entirely possible that stronglysignificant homologs could be obtained that fall outside of the rangesprovided.

Thus, a mutant FGF1 protein disclosed herein can have at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% sequence identityto SEQ ID NO: 5, but is not SEQ ID NO: 5 (which in some examples has oneor more, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the mutations ortruncations shown in Tables 1 and 2). In addition, exemplary mutatedFGF1 proteins have at least 80%, at least 85%, at least 90%, at least92%, at least 95%, at least 96%, at least 97%, at least 98% or at least99% sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 189, 191, 192, 193, 194, 195, 196,197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210,211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224,225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238,as well as such sequences schematically shown in FIGS. 23-26 (e.g., atleast 80%, at least 85%, at least 90%, at least 92%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% sequence identityto SEQ ID NO: 173, 174, 175, 177, 178, 179, 181, 182, 183, 185, 186,187, or 188), and retain the ability to reduce blood glucose levels invivo.

Similarly, exemplary mutated FGF1 coding sequences in some examples haveat least 70%, at least 80%, at least 85%, at least 90%, at least 92%, atleast 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO:18.

Similarly, exemplary β-Klotho-binding domain sequences that can be usedin the mutant FGF1 chimeras disclosed herein in some examples have atleast 70%, at least 80%, at least 85%, at least 90%, at least 92%, atleast 95%, at least 97%, at least 98%, or at least 99% sequence identityto SEQ ID NO: 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131,132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145,146 or β-Klotho-binding portions of SEQ ID NO: 168, 169, 170 or 171.

Similarly, exemplary FGFR1c binding sequences that can be used in themutant FGF1 chimeras disclosed herein in some examples have at least70%, at least 80%, at least 85%, at least 90%, at least 92%, at least95%, at least 97%, at least 98%, or at least 99% sequence identity toSEQ ID NO: 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158,159, 160, 161, 162, 163, 164, 165, 166, 167, or FGFR1c-binding portionsof SEQ ID NO: 168, 169, 170, 171, or multimers such as SEQ ID NO: 190.

Subject: Any mammal, such as humans, non-human primates, pigs, sheep,cows, dogs, cats, rodents and the like which is to be the recipient ofthe particular treatment, such as treatment with a mutated FGF1 proteinor chimera (or corresponding nucleic acid molecule) provided herein. Intwo non-limiting examples, a subject is a human subject or a murinesubject. In some examples, the subject has one or more metabolicdiseases, such as diabetes (e.g., type 2 diabetes, non-type 2 diabetes,type 1 diabetes, latent autoimmune diabetes (LAD), or maturity onsetdiabetes of the young (MODY)), polycystic ovary syndrome (PCOS),metabolic syndrome (MetS), obesity, non-alcoholic steatohepatitis(NASH), non-alcoholic fatty liver disease (NAFLD), dyslipidemia (e.g.,hyperlipidemia), cardiovascular disease (e.g., hypertension), orcombinations thereof. In some examples, the subject has elevated bloodglucose.

Transduced and Transformed: A virus or vector “transduces” a cell whenit transfers nucleic acid into the cell. A cell is “transformed” or“transfected” by a nucleic acid transduced into the cell when the DNAbecomes stably replicated by the cell, either by incorporation of thenucleic acid into the cellular genome, or by episomal replication.

Numerous methods of transfection are known to those skilled in the art,such as: chemical methods (e.g., calcium-phosphate transfection),physical methods (e.g., electroporation, microinjection, particlebombardment), fusion (e.g., liposomes), receptor-mediated endocytosis(e.g., DNA-protein complexes, viral envelope/capsid-DNA complexes) andby biological infection by viruses such as recombinant viruses {Wolff,J. A., ed, Gene Therapeutics, Birkhauser, Boston, USA (1994)}. In thecase of infection by retroviruses, the infecting retrovirus particlesare absorbed by the target cells, resulting in reverse transcription ofthe retroviral RNA genome and integration of the resulting provirus intothe cellular DNA.

Transgene: An exogenous gene supplied by a vector. In one example, atransgene includes a mutated FGF1 coding sequence (which may be part ofa chimera).

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector may include nucleic acidsequences that permit it to replicate in the host cell, such as anorigin of replication. A vector may also include one or more mutatedFGF1 coding sequences (which may be part of a chimera) and/or selectablemarker genes and other genetic elements known in the art. A vector cantransduce, transform or infect a cell, thereby causing the cell toexpress nucleic acids and/or proteins other than those native to thecell. A vector optionally includes materials to aid in achieving entryof the nucleic acid into the cell, such as a viral particle, liposome,protein coating or the like.

Overview

Provided herein are mutated FGF1 proteins that can include an N-terminaldeletion, one or more point mutations (such as amino acid substitutions,deletions, additions, or combinations thereof), or combinations ofN-terminal deletions and point mutations. Such mutated FGF1 proteins canbe part of a chimeric protein, such as a C-terminal portion of FGF21 or19 (e.g., SEQ ID NO: 86 or 100, respectively), a β-Klotho bindingprotein (e.g., SEQ ID NOS: 173, 174, 175, 177, 178, 179, 181, 182, 183,185, 186, and 187), or an FGFR1c binding protein (e.g., see SEQ ID NOS:188 and 189), or both a β-Klotho binding protein and an FGFR1c bindingprotein (e.g., linked directly or indirectly to any of SEQ ID NOS: 168,169, 170 or 171). Thus, when referring to a mutated FGF1 protein(s)herein, such reference also includes reference to mutated FGF1/FGF21,mutated FGF1/FGF19 chimeras, mutated FGF1/β-Klotho-binding chimeras,mutated FGF1/FGF1Rc-binding chimeras, or mutatedFGF1/3-Klotho-binding/FGF1Rc-binding chimeras.

Also provided are methods of using FGF1 mutant proteins orFGF1Rc-binding protein multimers (or their nucleic acid codingsequences) to lower glucose, for example to treat a metabolic disease.In some examples such methods include administering a therapeuticallyeffective amount of a mutated mature FGF21 protein or FGF1Rc-bindingprotein multimer to the mammal, or a nucleic acid molecule encoding themutated mature FGF21 protein or FGF1Rc-binding protein multimer or avector comprising the nucleic acid molecule, thereby reducing the bloodglucose, treating the one or more metabolic diseases, or combinationsthereof. Exemplary metabolic diseases that can be treated with thedisclosed methods include but are not limited to: type 2 diabetes,non-type 2 diabetes, type 1 diabetes, polycystic ovary syndrome (PCOS),metabolic syndrome (MetS), obesity, non-alcoholic steatohepatitis(NASH), non-alcoholic fatty liver disease (NAFLD), dyslipidemia (e.g.,hyperlipidemia), cardiovascular diseases (e.g., hypertension), latentautoimmune diabetes (LAD), or maturity onset diabetes of the young(MODY).

In some examples, mutations in FGF1 reduce the mitogenicity of maturewild-type FGF1 (e.g., SEQ ID NO: 5), such as a reduction of at least20%, at least 50%, at least 75% or at least 90%. For example, mutatedFGF1 can be mutated to decrease binding affinity for heparin and/orheparan sulfate compared to an FGF1 protein without the modification(e.g., a native or wild-type FGF1 protein). Methods of measuringmitogenicity are known in the art. In one example, the method providedin Example 2 is used.

In some examples, the mutant FGF1 protein is a truncated version of themature protein (e.g., SEQ ID NO: 5), which can include for exampledeletion of at least 5, at least 6, at least 9, at least 10, at least11, at least 12, at least 13, at least 14, at least 15, or at least 20consecutive N-terminal amino acids, such as the N-terminal 5 to 10, 5 to13, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acids of mature FGF1. In someexamples, such an N-terminally deleted FGF1 protein has reducedmitogenic activity as compared to wild-type mature FGF1 protein.

In some examples, one or more of the deleted N-terminal amino acids arereplaced with corresponding amino acids from FGF21 (e.g., see SEQ ID NO:20), such as at least 1, at least 2, at least 3, at least 4, at least 5,at least 10, at least 15, or at least 20 amino acids from FGF21, such as1-5, 1-4, 2-4, 4-6, 4-9, 3-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10corresponding amino acids from FGF21. An example of an FGF1 mutatedprotein with an N-terminal deletion having four corresponding N-terminalamino acids from FGF21 is shown in SEQ ID NO: 21 and 222. An example ofan FGF1 mutated protein with an N-terminal deletion having 16 N-terminalamino acids from FGF21 is shown in SEQ ID NO: 221. One skilled in theart will appreciate that amino acids from other FGFs besides FGF21 canbe used, including those having low affinity for FGFR4, including FGF3,FGF5, FGF7, FGF9 and FGF10. The N-terminal residues of FGF1 include anFGFR4 binding site, and FGFR4 signaling is associated with mitogenicactivity. In contrast, FGF21 has low affinity for FGFR4. Thus, replacingthe FGFR4 binding residues of FGF1 with those from FGF21 can be used toreduce mitogenicity of the resulting FGF1 mutant protein.

In some examples, mutations in FGF1 increase the thermostability ofmature or truncated FGF1 (e.g., SEQ ID NO: 5), such as an increase of atleast 20%, at least 50%, at least 75% or at least 90% compared to nativeFGF1. Exemplary mutations that can be used to increase thethermostability of mutated FGF1 include but are not limited to one ormore of: K12V, C117V, C117P, C117T, C117S, C117A and P134V (referred toas M1 mutations), L44F, C83T, C83S, C83A C83V, C117V, C117P, C117T,C117S, C117A and F132W (referred to as M2 mutations), and L44F, M67I,L73V, V109L, L111I, C117V, C117P, C117T, C117S, C117A A103G, R119G,R119V, Δ104-106, and Δ120-122 (referred to as M3 deletions), wherein thenumbering refers to SEQ ID NO: 5 (e.g., see Xia et al., PLoS One.7:e48210, 2012). For example, mutated FGF1 can be mutated to increasethe thermostability of the protein compared to an FGF1 protein withoutthe modification (e.g., SEQ ID NO: 5). Methods of measuringthermostability are known in the art. In one example, the methodprovided in Xia et al., PLoS One. 7:e48210, 2012 is used.

In some examples, the mutant FGF1 protein is a mutated version of themature protein (e.g., SEQ ID NO: 5), such as one containing at least 1,at least 4, at least 5, at least 6, at least 7, at least 8, at least 9,at least 10, at least 11, at least 12, at least 13, at least 14, atleast 15, at least 16, at least 17, at least 18, at least 19, at least20, at least 21, at least 22, at least 23, at least 24 or at least 25amino acid substitutions, such as 1-20, 1-10, 4-8, 5-25, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37 amino acidsubstitutions (such as those shown in Table 1). In some examples, themutant FGF1 protein includes deletion of one or more amino acids, suchas deletion of 1-10, 4-8, 5-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 amino acid deletions. In someexamples, the mutant FGF1 protein includes a combination of amino acidsubstitutions and deletions, such as at least 1 substitution and atleast 1 deletion, such as 1 to 10 substitutions with 1 to 10 deletions.

Exemplary mutations are shown in Table 1 below, with amino acidsreferenced to either SEQ ID NO: 2 or 5. One skilled in the art willrecognize that these mutations can be used singly, or in combination(such as 1-20, 1-10, 4-8, 5-25, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 41 of these amino acidsubstitutions and/or deletions). In addition, such mutant FGF1 proteinsare part of a chimeric protein, such as with FGF19, FGF21, a proteinthat selectively binds to β-Klotho, or a protein that selectively bindsto FGFR1c.

TABLE 1 Exemplary FGF1 mutations Location of Location of Point MutationPoint Mutation Position Position in in SEQ ID NO: 2 Mutation CitationSEQ ID NO: 5 K24 K9T K9 K25 K10T K10 K27 K12V K12 L29 L14A L14 Y30 Y15F,Y15A, Y15V Y15 C31 C16V, C16A, C16T, C16S C16 H36 H21Y H21 R50 R35E,R35V R35 Q55 Q40P Q40 L59 L44F L44 L61 L46V L46 S62 S47I S47 E64 E49Q,E49A E49 Y70 Y55F, Y55S, Y55A Y55 M82 M67I M67 L88 L73V L73 C98 C83T,C83S, C83A C83V C83 E102 E87V, E87A, E87S, E87T E87 H108 H93G, H93A H93Y109 Y94V, Y94F, Y94A Y94 N110 N95V, N95A, N95S, N95T N95 H117 H102YH102 A118 A103G A103 EKN 119-121 Δ104-106 EKN (104-106) F123 F108Y F108V124 V109L V109 L126 L111I L111 K127 K112D, K112E, K112Q K112 K128K113Q, K113E, K113D K113 C132 C117V, C117P, C117T, C117 C117S, C117AK133 K118N, K118E, K118V K118 R134 R119G, R119V, R119E R119 GPR 135-137Δ120-122 GPR (120-122) F147 F132W F132 L148 L133A, L133S L133 P149 P134VP134 L150 L135A, L135S L135

In some examples, the mutant FGF1 protein includes mutations at one ormore of the following positions: K9, K10, K12, L14, Y15, C16, H21, R35,Q40, L44, L46, S47, E49, Y55, M67, L73, C83, L86, E87, H93, Y94, N95,H102, A103, E104, K₁₀₅, N₁₀₆, F108, V109, L111, K₁₁₂, K₁₁₃, C117, K₁₁₈,R119, G120, P121, R122, F132, L133, P134, L135, such as one or more ofK₉, K₁₀, K₁₂, K₁₁₂, K₁₁₃, such as 1 to 5, 2 to 5, 3 to 6, 3 to 8, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41 or all 42 of these positions. In one example, K₉ and K₁₀ are replacedwith DQ (as in the mutated nuclear localization sequence) or withequivalent residues from FGF21 (or another FGF that does not bind toFGFR4) (wherein the numbering refers to SEQ ID NO: 5).

In some examples, the mutant FGF1 protein includes mutations at 1, 2, 3or 4 of the following positions: Y15, E87, Y94, and N95 (wherein thenumbering refers to SEQ ID NO: 5), such as one or more of Y15F, Y15A,Y15V, E87V, E87A, E87S, E87T, N95V, N95A, N95S, N95T, Y94V, Y94F, andY94A (such as 1, 2, 3 or 4 of these mutations). For example, E87 or N95can be replaced with a non-charged amino acid. In addition, Y15 and Y94can be replaced with an amino acid that destabilizes the hydrophobicinteractions. In some examples, the mutant FGF1 protein includesmutations on either side of Y15, E87, Y94, and N95, such as one or moreof L14, C16, H93, and T96, such as mutations at 1, 2, 3, or 4 of thesepositions.

In some examples, the mutant FGF1 protein includes mutations at 1, 2, 3,4, 5, 6, 7, 8, 9 or 10 of the following positions: Y15, C16, E87, H93,Y94, and N95 (wherein the numbering refers to SEQ ID NO: 5), such as oneor more of Y15F, Y15A, Y15V, E87V, E87A, E87S, E87T, H93A, N95V, N95A,N95S, N95T, Y94V, Y94F, and Y94A (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or10 of these mutations).

In some examples, the mutant FGF1 protein includes mutations at one ormore of the following positions: C16, C83, and C117 (wherein thenumbering refers to SEQ ID NO: 5), such as one or more of C16V, C16A,C16T, C16S, C83T, C83S, C83A C83V, C117V, C117P, C117T, C117S, and C117A(such as 1, 2, or 3 of these mutations).

In some examples, the mutant FGF1 protein includes mutations at only oneor two of the following positions: E87, Y94, and N95 (wherein thenumbering refers to SEQ ID NO: 5), such as one or two of E87V, E87A,E87S, E87T, Y94V, Y94F, Y94A, N95V, N95A, N95S, and N95T. In someexamples, the mutant FGF1 protein includes mutations at 1, 2, or 3 ofthe following positions: K₁₂, C83, and C117 (wherein the numberingrefers to SEQ ID NO: 5), such as one or more of K12V, K₁₂C, C83T, C83S,C83A, C83V, C117V, C117P, C117T, C117S, and C117A (such as 1, 2, or 3 ofthese mutations, such as K12V, C83T, and C117V).

FIG. 31 shows specific examples of positions that can be mutated in FGF1to alter its activity. For example, residues that interact with the FGF1receptor include Y15, E87, Y94 and N95. Thus, in some examples, 1, 2, 3,or 4 of these positions are mutated, for example the amino acid atposition 87 and/or 95 of SEQ ID NO: 5 can be changed to a V, A, S or T.In some examples, the amino acid at position 15 and/or 95 of SEQ ID NO:5 can be changed to a V, A, or F. In some examples, combinations ofthese changes are made.

FIG. 31 also shows that K₁₂ of FGF1 is predicted to be at the receptorinterface. Thus, K₁₂ of SEQ ID NO: 5 can be mutated, for example to a Vor C. FIG. 31 also shows that amino acids K₁₁₂, K₁₁₃, and K₁₁₈ are partof the heparin binding site, and thus can be mutated, for example to aE, Q, N, V or D, such as a N, E or V at position K₁₁₈, and a D, E or Qat positions K₁₁₂ and K₁₁₃. FIG. 31 also shows that amino acid R35 ofSEQ ID NO: 5 that forms a salt bridge with the D2 domain of the FGFreceptor, and thus can be mutated, for example to an E or V.

In some examples, the mutant FGF1 protein includes one or more of K12V,L46V, R35E, R35V, E87V, N95V, K₁₁₈N, K₁₁₈E, C117V, and P134V (whereinthe numbering refers to SEQ ID NO: 5). In some examples, the pointmutation includes replacing amino acid sequence ILFLPLPV (amino acids145-152 of SEQ ID NO: 2 and 4) to AAALPLPV (SEQ ID NO: 14), ILALPLPV(SEQ ID NO: 15), ILFAPLPV (SEQ ID NO: 16), or ILFLPAPA (SEQ ID NO: 17).In some examples, such an FGF1 protein with one or more point mutationshas reduced mitogenic activity as compared to wild-type mature FGF1protein. In some examples, the mutant FGF1 protein includes R35E(wherein the numbering refers to SEQ ID NO: 5).

In some examples, the mutant FGF1 protein includes at least 120consecutive amino acids from amino acids 5-141 of FGF1 (e.g., of SEQ IDNO: 2 or 4), (which in some examples can include further deletion ofN-terminal amino acids 1-20 and/or point mutations, such assubstitutions, deletions, or additions). In some examples, the mutantFGF1 protein includes at least 120 or at least 130 consecutive aminoacids from amino acids 5-141 of FGF1, such as at least 120 consecutiveamino acids from amino acids 5-141 of SEQ ID NO: 2 or 4 or at least 120consecutive amino acids from SEQ ID NO: 5.

In some examples, the mutant FGF1 protein includes both an N-terminaltruncation and point mutations. Specific exemplary FGF1 mutant proteinsare shown in SEQ ID NOS: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 113, 114, 115, 116, 117, 118, 119, 120, 191, 192,193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 225, 226,227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 and 238. In someexamples, the FGF1 mutant includes an N-terminal deletion, but retains amethionine at the N-terminal position. In some examples, the FGF1 mutantis 120-140 or 125-140 amino acids in length.

In some examples, the FGF1 mutant protein is part of a chimeric protein.For example, one end of the mutant FGF1 mutant protein can be joineddirectly or indirectly to the end of FGF19 or FGF21, such as aC-terminal region of FGF 19 or FGF21. In some examples, the mutated FGF1portion of the chimera is at the N-terminus of the chimera, and theFGF19 or FGF21 portion is the C-terminus of the chimera. However, thiscan be reversed, such that the mutated FGF1 portion of the chimera isthe C-terminus of the chimera, and the FGF19 or FGF21 portion is theN-terminus of the chimera. For example, at least 10, at least 20, atleast 30, at least 40, at least 41, at least 42, at least 43, at least44, at least 45, at least 46, at least 47, at least 48, at least 49, atleast 50 or at least 60 C-terminal amino acids of FGF19 or FGF21 (suchas the C-terminal 60, 55, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40,35, 30, 25, 20, 15 or 10 amino acids) can be part of the chimera.Examples of C-terminal fragments of FGF21 and FGF19 that can be used areshown in SEQ ID NOS: 86 and 100, respectively. In some examples, themutant FGF1 and FGF21 or FGF19 portion are linked indirectly through theuse of a linker, such as one composed of at least 5, at least 10, atleast 15 or at least 20 amino acids. In one example the linker is a polyalanine.

In some examples, the FGF1 mutant protein is part of a chimeric proteinwith a β-Klotho-binding protein. For example, one end of the mutant FGF1mutant protein can be joined directly or indirectly to the end of aβ-Klotho-binding protein (see for example FIGS. 23-26). In someexamples, the mutated FGF1 portion of the chimera is at the N-terminusof the chimera, and the β-Klotho-binding protein portion is theC-terminus of the chimera (e.g., see FIGS. 23B-23D, 23G-231 and 25B-25D,25G-25I, respectively). However, this can be reversed, such that themutated FGF1 portion of the chimera is the C-terminus of the chimera,and the β-Klotho binding protein portion is the N-terminus of thechimera (e.g., see FIGS. 24B-24D, 24F-24H and 26B-26D, 26F-26H).Examples of β-Klotho-binding proteins that can be used are shown in SEQID NOS: 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133,134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145 and 146 andβ-Klotho-binding portions of SEQ ID NOS: 168, 169, 170, and 171. In someexamples, the mutant FGF1 and β-Klotho-binding protein portion arelinked indirectly through the use of a linker, such as one composed ofat least 5, at least 10, at least 15 or at least 20 amino acids. In oneexample the linker is a poly alanine.

In some examples, the FGF1 mutant protein is part of a chimeric proteinwith an FGFR1c-binding protein. For example, one end of the mutant FGF1mutant protein can be joined directly or indirectly to the end of anFGFR1c-binding protein. In some examples, the mutated FGF1 portion ofthe chimera is at the N-terminus of the chimera, and the FGFR1c-bindingprotein portion is the C-terminus of the chimera (e.g., see FIG. 23J).However, this can be reversed, such that the mutated FGF1 portion of thechimera is the C-terminus of the chimera, and the FGFR1c-binding proteinportion is the N-terminus of the chimera (e.g., see FIG. 241). Examplesof FGFR1c-binding proteins that can be used are shown in SEQ ID NOS:147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166, and 167 and FGFR1c-binding portions of168, 169, 170 and 171. In some examples, the mutant FGF1 andFGFR1c-binding protein portion are linked indirectly through the use ofa linker, such as one composed of at least 5, at least 10, at least 15or at least 20 amino acids. In one example the linker is a poly alanine.

In some examples, the FGF1 mutant protein is part of a chimeric proteinwith both an FGFR1c-binding protein and a β-Klotho-binding protein, inany order. For example, one end of the mutant FGF1 mutant protein can bejoined directly or indirectly to the end of anFGFR1c-binding/β-Klotho-binding or β-Klotho-binding/FGFR1c-bindingchimeric protein. In some examples, the mutated FGF1 portion of thechimera is at the N-terminus of the chimera, and theFGFR1c-binding/β-Klotho-binding or 13-Klotho-binding/FGFR1c-bindingchimeric protein portion is the C-terminus of the chimera (e.g., seeFIG. 23K). However, this can be reversed, such that the mutated

FGF1 portion of the chimera is the C-terminus of the chimera, and theFGFR1c-binding/β-Klotho-binding or β-Klotho-binding/FGFR1c-bindingchimeric protein portion is the N-terminus of the chimera (e.g., seeFIG. 24J). In one example the FGFR1c-binding/β-Klotho-binding orβ-Klotho-binding/FGFR1c-binding chimeric protein is any one of thoseshown in SEQ ID NOS: 168, 169, 170, and 171. In some examples, themutant FGF1 and FGFR1c-binding/β-Klotho-binding orβ-Klotho-binding/FGFR1c-binding chimeric protein portion are linkedindirectly through the use of a linker, such as one composed of at least5, at least 10, at least 15 or at least 20 amino acids. In one examplethe linker is a poly alanine. In some examples, the FGF1 mutant proteinor chimera including such includes at least 80% sequence identity to SEQID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 173, 174, 175, 177, 178, 179, 181, 182, 183, 185, 186, 187,188, 189, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202,203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230,231, 232, 233, 234, 235, 236, 237 or 238. Thus, the FGF1 mutant proteincan have at least 90%, at least 95%, at least 96%, at least 97%, atleast 98% or at least 99% sequence identity to SEQ ID NO: 6, 7, 8, 9,10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 173, 174,175, 177, 178, 179, 181, 182, 183, 185, 186, 187, 188, 189, 191, 192,193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220,221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,235, 236, 237 or 238 (but is not a native FGF1 sequence, such as SEQ IDNO: 5). In some examples, the FGF1 mutant protein includes or consistsof SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101, 102,103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116,117, 118, 119, 120, 173, 174, 175, 177, 178, 179, 181, 182, 183, 185,186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200,201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214,215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228,229, 230, 231, 232, 233, 234, 235, 236, 237 and 238. The disclosureencompasses variants of the disclosed FGF1 mutant proteins, such as SEQID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 173, 174, 175, 177, 178, 179, 181, 182, 183, 185, 186, 187,188, 189, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202,203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230,231, 232, 233, 234, 235, 236, 237 or 238 having 1 to 8, 2 to 10, 1 to 5,1 to 6, or 5 to 10 mutations, such as conservative amino acidsubstitutions.

In some examples, a mutant FGF1/FGF21 chimera protein includes at least80% sequence identity to SEQ ID NO: 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 219, 221, 222, or 223. Thus, the mutant FGF1/FGF21 chimericprotein can have at least 90%, at least 95%, at least 96%, at least 97%,at least 98% or at least 99% sequence identity to SEQ ID NO: 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 219, 221, 222, or 223. In someexamples, the mutant FGF1/FGF21 chimera protein includes or consists ofSEQ ID NO: 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 219, 221,222, or 223. The disclosure encompasses variants of the disclosed mutantFGF1/FGF21 chimera proteins, such as SEQ ID NO: 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 219, 221, 222, or 223 having 1-8 mutations, suchas conservative amino acid substitutions.

In some examples, a mutant FGF1/FGF19 chimera protein includes at least80% sequence identity to SEQ ID NO: 101, 102, 103, 104, 105, 106, 107,108, 109, 110, 111, 112, 220, or 224. Thus, the mutant FGF1/FGF19chimeric protein can have at least 90%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 220, or 224.In some examples, the mutant FGF1/FGF19 chimera protein includes orconsists of any of SEQ ID NOS: 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 220, or 224. The disclosure encompasses variants ofthe disclosed mutant FGF1/FGF19 chimera proteins, such as SEQ ID NO:101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 220, or 224having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations, such as conservativeamino acid substitutions.

In some examples, a mutant FGF1/β-Klotho-binding protein chimeraincludes at least 80% sequence identity to SEQ ID NO: 173, 174, 175,177, 178, 179, 181, 182, 183, 185, 186, or 187. Thus, the mutantFGF1/β-Klotho chimeric protein can have at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% sequence identityto SEQ ID NO: 1173, 174, 175, 177, 178, 179, 181, 182, 183, 185, 186, or187. In some examples, the mutant FGF1/β-Klotho chimera protein includesor consists of SEQ ID NO: 173, 174, 175, 177, 178, 179, 181, 182, 183,185, 186, or 187. The disclosure encompasses variants of the disclosedmutant FGF1/β-Klotho chimera proteins, such as SEQ ID NO: 173, 174, 175,177, 178, 179, 181, 182, 183, 185, 186, or 187 having 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 mutations, such as conservative amino acid substitutions.

In some examples, a mutant FGF1/FGF1Rc-binding protein chimera includesat least 80% sequence identity to any of SEQ ID NOS: 188-189. Thus, themutant FGF1/FGF1Rc chimeric protein can have at least 90%, at least 95%,at least 96%, at least 97%, at least 98% or at least 99% sequenceidentity to SEQ ID NO: 188 or 189. In some examples, the mutantFGF1/FGF1Rc chimera protein includes or consists of any of SEQ ID NOS:188-189. The disclosure encompasses variants of the disclosed mutantFGF1/FGF1Rc chimera proteins, such as SEQ ID NO: 188 or 189 having 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 mutations, such as conservative amino acidsubstitutions.

In one example the FGFR1c-binding/β-Klotho-binding orβ-Klotho-binding/FGFR1c-binding protein portion of a chimera includes atleast 80% sequence identity to SEQ ID NO: 168, 169, 170 or 171, such asat least 90%, at least 95%, at least 96%, at least 97%, at least 98% orat least 99% sequence identity to SEQ ID NO: 168, 169, 170 or 171.

In one example an FGFR1c-binding protein multimer includes at least onemonomer having 80% sequence identity to the FGFR1c-binding portion ofSEQ ID NO: 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158,159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, or 170, such asat least 90%, at least 95%, at least 96%, at least 97%, at least 98% orat least 99% sequence identity to the FGFR1c portion of SEQ ID NO: 147,148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161,162, 163, 164, 165, 166, 167, 168, 169, or 170. In one example anFGFR1c-binding protein dimer includes at least 80% sequence identity SEQID NO: 190, such as at least 90%, at least 95%, at least 96%, at least97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 190.

Also provided are isolated nucleic acid molecules encoding the disclosedmutated FGF1 proteins and chimeras, such as a nucleic acid moleculeencoding a protein having at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO:6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105,106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 173, 174, 175, 177, 178, 179, 181, 182, 183, 185, 186, 187, 188,189, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203,204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217,218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231,232, 233, 234, 235, 236, 237, or 238 (but is not a native FGF1sequence). One exemplary coding sequence is shown in SEQ ID NO: 18;thus, the disclosure provides sequences having at least 80%, at least85%, at least 90%, at least 95%, at least 98%, or at least 99% sequenceidentity to any of SEQ ID NO: 18. Vectors and cells that include suchnucleic acid molecules are also provided. For example, such nucleic acidmolecules can be expressed in a host cell, such as a bacterium or yeastcell (e.g., E. coli), thereby permitting expression of the mutated FGF1protein. The resulting mutated FGF1 protein can be purified from thecell.

Methods of using the disclosed mutated FGF1 proteins and chimeras (ornucleic acid molecules encoding such), as well as the FGFR1c-bindingprotein multimers, are provided. As discussed herein, the mutated matureFGF1 protein can include a deletion of at least six contiguousN-terminal amino acids, at least one point mutation, or combinationsthereof. For example, such methods include administering atherapeutically effective amount of a disclosed mutated FGF1 protein orchimeric protein including the mutant FGF1 mutant protein, orFGFR1c-binding protein multimer, (such as at least 0.01, at least 0.1mg/kg, or at least 0.5 mg/kg) (or nucleic acid molecules encoding such)to reduce blood glucose in a mammal, such as a decrease of at least 5%,at least 10%, at least 25% or at least 50%, for example as compared toadministration of no mutant FGF1 mutant protein or FGFR1c-bindingprotein multimer (e.g., administration of PBS).

In one example, the method is a method of reducing fed and fasting bloodglucose, improving insulin sensitivity and glucose tolerance, reducingsystemic chronic inflammation, ameliorating hepatic steatosis in amammal, reducing triglycerides, decreasing insulin resistance, reducinghyperinsulinemia, increasing glucose tolerance, reducing hyperglycemia,reducing food intake, or combinations thereof. Such a method can includeadministering a therapeutically effective amount of a disclosed mutatedFGF1 protein or chimeric protein including the mutant FGF1 mutantprotein, or FGFR1c-binding protein multimer, (such as at least 0.5mg/kg) (or nucleic acid molecules encoding such) to reduce fed andfasting blood glucose, improve insulin sensitivity and glucosetolerance, reduce systemic chronic inflammation, ameliorate hepaticsteatosis in a mammal, reduce food intake, or combinations thereof.

In one example, the method is a method of treating a metabolic disease(such as metabolic syndrome, diabetes, or obesity) in a mammal. Such amethod can include administering a therapeutically effective amount of adisclosed mutated FGF1 protein or chimeric protein including the mutantFGF1 mutant protein, or FGFR1c-binding protein multimer, (such as atleast 0.5 mg/kg) (or nucleic acid molecules encoding such) to treat themetabolic disease.

In some examples, the mammal, such as a human, cat or dog, has diabetes.Methods of administration are routine, and can include subcutaneous,intraperitoneal, intramuscular, or intravenous injection.

In some examples, use of the FGF1 mutants or chimeric proteins includinga mutant FGF1 mutant protein, or FGFR1c-binding protein multimer,disclosed herein does not lead to (or significantly reduces, such as areduction of at least 20%, at least 50%, at least 75%, or at least 90%)the adverse side effects observed with thiazolidinediones (TZDs)therapeutic insulin sensitizers, including weight gain, increased liversteatosis and bone fractures (e.g., reduced affects on bone mineraldensity, trabecular bone architecture and cortical bone thickness).

Provided are methods of reducing fed and fasting blood glucose,improving insulin sensitivity and glucose tolerance, reducing systemicchronic inflammation, ameliorating hepatic steatosis, reducing foodintake, or combinations thereof, in a mammal. Such methods can includeadministering a therapeutically effective amount of a FGF1 mutant and/orFGFR1c-binding protein multimer disclosed herein, including those thatfurther include a β-Klotho-binding peptide and/or FGFR1c-bindingpeptide, to the mammal, or a nucleic acid molecule encoding the FGF1mutant or multimer or a vector comprising the nucleic acid molecule,thereby reducing fed and fasting blood glucose, improving insulinsensitivity and glucose tolerance, reducing systemic chronicinflammation, ameliorating hepatic steatosis, reduce one or more non-HDLlipid levels, reduce food intake, or combinations thereof, in a mammal.In some examples, the fed and fasting blood glucose is reduced in thetreated subject by at least 10%, at least 20%, at least 30%, at least50%, at least 75%, or at least 90% as compared to an absence ofadministration of the FGF1 mutant and/or FGFR1c-binding proteinmultimer. In some examples, insulin sensitivity and glucose tolerance isincreased in the treated subject by at least 10%, at least 20%, at least30%, at least 50%, at least 75%, or at least 90% as compared to anabsence of administration of the FGF1 mutant and/or FGFR1c-bindingprotein multimer. In some examples, systemic chronic inflammation isreduced in the treated subject by at least 10%, at least 20%, at least30%, at least 50%, at least 75%, or at least 90% as compared to anabsence of administration of the FGF1 mutant and/or FGFR1c-bindingprotein multimer. In some examples, hepatic steatosis is reduced in thetreated subject by at least 10%, at least 20%, at least 30%, at least50%, at least 75%, or at least 90% as compared to an absence ofadministration of the FGF1 mutant and/or FGFR1c-binding proteinmultimer. In some examples, one or more lipids (such as a non-HDL, forexample IDL, LDL and/or VLDL) are reduced in the treated subject by atleast 10%, at least 20%, at least 30%, at least 50%, at least 75%, or atleast 90% as compared to an absence of administration of the FGF1 mutantand/or FGFR1c-binding multimer In some examples, triglyceride and orcholesterol levels are reduced with the FGF1 mutant and/orFGFR1c-binding protein multimer by at least 10%, at least 20%, at least30%, at least 50%, at least 75%, or at least 90% as compared to anabsence of administration of the FGF1 mutant and/or FGFR1c-bindingprotein multimer. In some examples, the amount of food intake is reducedin the treated subject by at least 10%, at least 20%, at least 30%, atleast 50%, at least 75%, or at least 90% as compared to an absence ofadministration of the FGF1 mutant and/or FGFR1c-binding protein multimer(such as within 12 hours, within 24 hours, or within 48 hours of thetreatment, such as within 12 to 24 hours, within 12 to 36 hours, orwithin 24 to 48 hours). In some examples, combinations of thesereductions are achieved.

Mutated FGF1 Proteins

The present disclosure provides mutated FGF1 proteins that can includean N-terminal deletion, one or more point mutations (such as amino acidsubstitutions, deletions, additions, or combinations thereof), orcombinations of N-terminal deletions and point mutations. Such proteinsand corresponding coding sequences can be used in the methods providedherein. In some examples, the disclosed FGF1 mutant proteins havereduced mitogenicity compared to mature native FGF1 (e.g., SEQ ID NO:5), such as a reduction of at least 20%, at least 50%, at least 75% orat least 90%. For example, mutated FGF1 can be mutated to decreasebinding affinity for heparin and/or heparan sulfate compared to a nativeFGF1 protein without the modification. Methods of measuring mitogenicityare known in the art.

In some examples, the mutant FGF1 protein is a truncated version of themature protein (e.g., SEQ ID NO: 5), which can include for exampledeletion of at least 5, at least 6, at least 9, at least 10, at least11, at least 12, at least 13, at least 14, at least 15, or at least 20consecutive N-terminal amino acids. Thus, in some examples, the mutantFGF1 protein is a truncated version of the mature protein (e.g., SEQ IDNO: 5), such a deletion of the N-terminal 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19 or 20 amino acids shown in SEQ ID NO: 5. Examplesof N-terminally truncated FGF1 proteins are shown in SEQ ID NOS: 6, 7,8, 9, 21, 24, 25, 26, 27, 32, 33, 34, 35, 36, 37, 38, 39, 44, 45, 46,47, 48, 49, 50, 51, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 71, 72, 74,75, 76, 77, 79, 80, 81, 82, 194, 195, 197, 198, 202, 203, 205, 206, 214,215, 216, 217, 221, 222, 225, 228, 232, and 238. In some examples, theFGF1 mutant includes an N-terminal deletion, but retains a methionine atthe N-terminal position. In some examples, such an N-terminally deletedFGF1 protein has reduced mitogenic activity as compared to wild-typemature FGF1 protein.

In some examples, one or more of the deleted N-terminal amino acids arereplaced with corresponding amino acids from FGF21 (e.g., see SEQ ID NO:20), such as at least 1, at least 2, at least 3, at least 4, at least 5,at least 10, at least 15, or at least 20 amino acids from FGF21, such as1-5, 1-4, 2-4, 4-6, 4-9, 3-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10corresponding amino acids from FGF21. An example of an FGF1 mutatedprotein with an N-terminal deletion having four corresponding N-terminalamino acids from FGF21 is shown in SEQ ID NO: 21. The N-terminalresidues of FGF1 include an FGFR4 binding site, and FGFR4 signaling isassociated with mitogenic activity. In contrast, FGF21 has low affinityfor FGFR4. Thus, replacing the FGFR4 binding residues of FGF1 with thosefrom FGF21 (or from another FGF having low affinity for FGFR4, includingFGF3, FGF5, FGF7, FGF9 and FGF10) can be used to reduce mitogenicity ofthe resulting FGF1 mutant protein.

Thus, in some examples, the mutant FGF1 protein includes at least 120consecutive amino acids from amino acids 5-141 or 5-155 of FGF1 (e.g.,of SEQ ID NO: 2 or 4), (which in some examples can include furtherdeletion of N-terminal amino acids 1-20 and/or point mutations, such assubstitutions, deletions, or additions). In some examples, the mutantFGF1 protein includes at least 120 consecutive amino acids from aminoacids 1-140 of FGF1 (e.g., of SEQ ID NO: 5), (which in some examples caninclude further deletion of N-terminal amino acids 1-20 and/or pointmutations, such as substitutions, deletions, or additions). Thus, insome examples, the mutant FGF1 protein includes at least 120 consecutiveamino acids from amino acids 5-141 of FGF1, such as at least 120, atleast 121, at least 122, at least 123, at least 124, at least 125, atleast 126, at least 127, at least 128, at least 129, at least 130, atleast 131, at least 132, at least 133, at least 134, at least 135, atleast 136, at least 137, at least 138, at least 139, or at least 140consecutive amino acids from amino acids 5-141 of SEQ ID NO: 2 or 4(such as 120-130, 120-135, 130-135, 130-140, or 120-140 consecutiveamino acids from amino acids 5-141 of SEQ ID NO: 2 or 4). In someexamples, the mutant FGF1 protein includes at least 120 or at least 130consecutive amino acids from amino acids 5-141 of FGF1, such as at least120 consecutive amino acids from amino acids 5-141 of SEQ ID NO: 2 or 4or at least 120 consecutive amino acids from SEQ ID NO: 5. Thus, in someexamples, the mutant FGF1 protein includes at least 120, at least 121,at least 122, at least 123, at least 124, at least 125, at least 126, atleast 127, at least 128, at least 129, at least 130, at least 131, atleast 132, at least 133, at least 134, at least 135, at least 136, atleast 137, at least 138, at least 139, or at least 140 consecutive aminoacids from SEQ ID NO: 5 (such as 120-130, 120-135, or 120-140consecutive amino acids from SEQ ID NO: 5). Examples of least 120consecutive amino acids from amino acids 5 to 141 of FGF1 that can beused to generate a mutant FGF1 protein includes but are not limited toamino acids 4 to 140 of SEQ ID NO: 5 and the protein sequence shown inany of SEQ ID NOs: 6, 7, 8, and 9.

In some examples, the mutant FGF1 protein is a mutated version of themature protein (e.g., SEQ ID NO: 5), or a N-terminal truncation of themature protein (e.g., SEQ ID NOS: 7, 8, 9), such as one containing atleast 1, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, at least 15, at least 16, at least 17, at least 18, at least 19, orat least 20 amino acid substitutions, such as 1-20, 1-10, 4-8, 5-12,5-10, 5-25, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 aminoacid substitutions. For example, point mutations can be introduced intoan FGF1 sequence to decrease mitogenicity, increase stability, decreasebinding affinity for heparin and/or heparan sulfate (compared to theportion of a native FGF1 protein without the modification), orcombinations thereof. Specific exemplary point mutations that can beused are shown above in Table 1, and exemplary combinations are providedin FIGS. 1, 3A-3D, 4A-4B, 5A-5B, 6A-6B, 20, and 27-30.

In some examples, the mutant FGF1 protein includes mutations (such as asubstitution or deletion) at one or more of the following positions K9,K10, K12, L14, Y15, C16, H21, R35, Q40, L44, L46, S47, E49, Y55, M67,L73, C83, L86, E87, H93, Y94, N95, H102, A103, E104, K105, N106, F108,V109, L111, K112, K113, C117, K118, R119, G120, P121, R122, F132, L133,P134, L135, such as one or more of K9, K10, K12, K112, K113, such as 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41 or all 42 of these positions. In some examples the mutant FGF1protein has as one or more of K9T, K10T, K12V, L14A, Y15F, Y15A, Y15V,C16V, C16A, C16T, C16S, H21Y, R35E, R35V, Q40P, L44F, L46V, S47I, E49Q,E49A, Y55F, Y55S, Y55A, M67I, L73V, C83T, C83S, C83A C83V, E87V, E87A,E87S, E87T, H93G, H93A, Y94V, Y94F, Y94A, N95V, N95A, N95S, N95T, H102Y,A103G, 4104-106, F108Y, V109L, L111I, K112D, K112E, K112Q, K113Q, K113E,K113D, C117V, C117P, C117T, C117S, C117A, K118N, K118E, K118V, R119G,R119V, R119E, Δ120-122, F132W, L133A, L133S, P134V, L135A, L135S,(wherein the numbering refers to SEQ ID NO: 5), such as 1 to 5, 1 to 10,2 to 5, 2 to 10, 2 to 20, 5 to 10, 5 to 40, or 5 to 20 of thesemutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20 of these mutations.

In some examples, the mutant FGF1 protein includes one or more (such as2, 3, 4, 5 or 6) of K12V, R35E, R35V, L46V, E87V, N95V, C117V/A, K118N,K118E/V, and P134V (wherein the numbering refers to SEQ ID NO: 5). Insome examples, the point mutation includes replacing amino acid sequenceILFLPLPV (amino acids 145-152 of SEQ ID NO: 2 and 4) to AAALPLPV (SEQ IDNO: 14), ILALPLPV (SEQ ID NO: 15), ILFAPLPV (SEQ ID NO: 16), or ILFLPAPA(SEQ ID NO: 17). In some examples, such an FGF1 protein with one or morepoint mutations has reduced mitogenic activity as compared to wild-typemature FGF1 protein. In some examples, the mutant FGF1 protein includesR35E, (wherein the numbering refers to SEQ ID NO: 5). Examples of FGF1mutant proteins containing point mutations include but are not limitedto the protein sequence shown in SEQ ID NOS: 10, 11, 12, 13, 22, 23, 28,29, 30, 31, 40, 41, 42, 43, 42, 53, 54, 55, 56, 67, 68, 69, 70, 73, 78,83, 84, 113, 114, 115, 116, 117, 118, 119, 120, 191, 192, 193, 196, 199,200, 201, 204, 207, 208, 209, 210, 211, 212, 213, 218, 226, 227, 229,230, 231, 232, 233, 234, 235, 236, and 237.

In some examples, mutations in FGF1 increase the thermostability ofmature or truncated native FGF1. For example, mutations can be made atone or more of the following positions. Exemplary mutations that can beused to increase the thermostability of mutated FGF1 include but are notlimited to one or more of: K12, C117, P134, L44, C83, F132, M67, L73,V109, L111, A103, R119, Δ104-106, and Δ120-122, Q40, H93, S47, whereinthe numbering refers to SEQ ID NO: 5 (e.g., see Xia et al., PLoS One.7:e48210, 2012). In some examples, thermostability of FGF1 is increasedby using one or more of the following mutations: Q40P and S471 or Q40P,S47I, and H93G (or any other combination of these mutations).

In some examples, the FGF1 mutant protein is part of a chimeric protein.For example, any mutant FGF1 protein provided herein can be joineddirectly or indirectly to the end of a β-Klotho-binding protein, anFGFR1c binding protein, both a β-Klotho-binding protein and an FGFR1cbinding protein, FGF19, or FGF21, such as a C-terminal region of FGF 19or FGF21. For example, at least 10, at least 20, at least 30, at least40, at least 41, at least 42, at least 43, at least 44, at least 45, atleast 46, at least 47, at least 48, at least 49, at least 50 or at least60 C-terminal amino acids of FGF19 or FGF21 (such as the C-terminal 60,55, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 35, 30, 25, 20, 15 or 10amino acids) can be part of the chimera. Examples of C-terminalfragments of FGF21 and FGF19 that can be used are shown in SEQ ID NOS:86 and 100, respectively. Examples of β-Klotho-binding proteins that canbe used are shown in SEQ ID NOS: 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145 and 146. Examples of FGFR1c-binding proteins that can beused are shown in SEQ ID NOS: 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, and 167.Examples of β-Klotho-binding/FGFR1c-binding protein chimeras that can bedirectly or indirectly attached to a mutant FGF1 protein are shown inSEQ ID NOS: 168, 169, 170, and 171.

In some examples, the mutant FGF1 protein includes both an N-terminaltruncation and point mutations. Specific exemplary FGF1 mutant proteinsare shown in SEQ ID NOS: 6-13, 21-84, 113-120, 191-218 and 225-238. Insome examples, the FGF1 mutant protein includes at least 80% sequenceidentity to any of SEQ ID NOS: 6-13, 21-84, 113-120, 191-218 and225-238. Thus, the FGF1 mutant protein can have at least 90%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% sequenceidentity to any of SEQ ID NOS: 6-13, 21-84, 113-120, 191-218 and225-238. In some examples, the FGF1 mutant protein includes or consistsof any of SEQ ID NOS: 6-13, 21-84, 113-120, 191-218 and 225-238. Thedisclosure encompasses variants of the disclosed FGF1 mutant proteins,such as any of SEQ ID NOS: 6-13, 21-84, and 113-120, 191-218 and 225-238having 1 to 20, 1 to 15, 1 to 10, 1 to 8, 2 to 10, 1 to 5, 1 to 6, 2 to12, 3 to 12, 5 to 12, or 5 to 10 mutations, such as conservative aminoacid substitutions. Such mutant FGF1 proteins can be used to generate anFGF1 mutant chimera.

In some examples, the mutant FGF1 protein has at its N-terminus amethionine. In some examples, the mutant FGF1 protein is at least 120amino acids in length, such as at least 125, at least 130, at least 135,at least 140, at least 145, at least 150, at least 155, at least 160, orat least 175 amino acids in length, such as 120-160, 125-160, 130-160,150-160, 130-200, 130-180, 130-170, or 120-160 amino acids in length.

Exemplary N-terminally truncated FGF1 sequences and FGF1 point mutationsthat can be used to generate an FGF1 mutant protein are shown in Tables1 and 2 (as well as those provided in any of SEQ ID NOS: 6, 7, 8, 9, 10,11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 173, 174, 175,177, 178, 179, 181, 182, 183, 185, 186, 187, 188, 189, 191, 192, 193,194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207,208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235,236, 237 and 238). One skilled in the art will appreciate that anyN-terminal truncation in Table 2 (as well as those provided in any ofSEQ ID NOS: 6, 7, 8, 9, 21, 24, 25, 26, 27, 32, 33, 34, 35, 36, 37, 38,39, 44, 45, 46, 47, 48, 49, 50, 51, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 71, 72, 74, 75, 76, 77, 79, 80, 81, 82, 194, 195, 197, 198, 202,203, 205, 206, 214, 215, 216, 217, 221, 222, 225, 228, 232, and 238) canbe combined with any FGF1 point mutation in Table 1 or Table 2, togenerate an FGF1 mutant protein, and that such an FGF1 mutant proteincan be used directly or be used as part of a mutantFGF1/β-Klotho-binding protein chimera, mutant FGF1/FGFR1c-bindingprotein chimera, mutant FGF1/β-Klotho-binding protein/FGFR1c-bindingprotein chimera, mutant FGF1/FGF21 or mutant FGF1/FGF19 chimera. Inaddition, mutations can be made to the sequences shown in the Table,such as one or more of the mutations discussed herein (such as 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 aminoacid substitutions, such as conservative amino acid substitutions,deletions, or additions).

TABLE 2 Exemplary mutations that can be used togenerate an FGF1 mutant protein FGF1 Point Mutations FGF1 FragmentsFNLPPGNYKK PVLLYCSNGG PPGNYK     KPKLLYCSNG HFLRILPDGT VDGTRDRSDQGHFLRILPDG TVDGTRDRSD HIQLQLSAES VGEVYIKSTE QHIQLQLSAE SVGEVYIKSTTGQYLAMDTD GLLYGSQTPN ETGQYLAMDT DGLLYGSQTP EECLFLERLE ENHYVTYISKNEECLFLERL EENHYNTYIS KHAEKNWFVG LKKNGSCKRG KKHAEKNWFV GLKKNGSCKRPRTHYGQKAI LFLPLPVSSD GPRTHYGQKA ILFLPLPVSSD (SEQ ID NO: 10)(SEQ ID NO: 6) FNLPPGNYKK PVLLYCSNGG KPKLLYCSNGG HFLRILPDGTHFLRILPDGT VDGTRDRSDQ VDGTRDRSDQ HIQLQLSAES HIQLQVSAES VGEVYIKSTEVGEVYIKSTE TGQYLAMDTD TGQYLAMDTD GLLYGSQTPN GLLYGSQTPN EECLFLERLEEECLFLVRLE ENHYVTYISK ENHYNTYISK KHAEKNWFVG KHAEKNWFVG LKKNGSCKRGLKKNGSCKRG PRTHYGQKAI PRTHYGQKAI LFLVLPVSSD LFLPLPVSSD (SEQ ID NO: 11)(SEQ ID NO: 7) NYKK       PKLLYCSNGG LYCSNGG    HFLRILPDGTHFLRILPDGT VDGTRDRSDQ VDGTRDRSDQ HIQLQLSAES HIQLQLSAES VGEVYIKSTEVGEVYIKSTE TGQYLAMDTD TGQYLAMDTD GLLYGSQTPN GLLYGSQTPN EECLFLERLEEECLFLERLE ENHYNTYISK ENHYNTYISK KHAEKNWFVG KHAEKNWFVG LKKNGSCNRGLKKNGSCKRG PRTHYGQKAI PRTHYGQKAI LFLPLPVSSD LFLPLPVSSD (SEQ ID NO: 12)(SEQ ID NO: 8) NYKK       PKLLYCSNGG KLLYCSNGG  HFLRILPDGTHFLRILPDGT VDGTRDRSDQ VDGTRDRSDQ HIQLQLSAES HIQLQLSAES VGEVYIKSTEVGEVYIKSTE TGQYLAMDTD TGQYLAMDTD GLLYGSQTPN GLLYGSQTPN EECLFLERLEEECLFLERLE ENHYNTYISK ENHYNTYISK KHAEKNWFVG KHAEKNWFVG LKKNGSCERGLKKNGSCKRG PRTHYGQKAI PRTHYGQKAI LFLPLPVSSD LFLPLPVSSD (SEQ ID NO: 13)(SEQ ID NO: 9) GGQVKPKLLYCSNG GHFLRILPDG TVDGTRDRSD QHIQLQLSAESVGEVYIKST ETGQYLAMDT DGLLYGSQTP NEECLFLERL EENHYNTYIS KKHAEKNWFVGLKKNGSCKR GPRTHYGQKA ILFLPLPVSSD (SEQ ID NO: 21)

Exemplary mutant FGF1 proteins are provided in SEQ ID NOS: 6, 7, 8, 9,10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 113, 114, 115,116, 117, 118, 119, 120, 191, 192, 193, 194, 195, 196, 197, 198, 199,200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,214, 215, 216, 217, 218, 225, 226, 227, 228, 229, 230, 231, 232, 233,234, 235, 236, 237 and 238, mutant FGF1/FGF21 chimeras in SEQ ID NOS:87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 219, 221, 222 and 223,mutant FGF1/FGF19 chimeras in SEQ ID NOS: 101, 102, 103, 104, 105, 106,107, 108, 109, 110, 111, 112, 220 and 224, mutant FGF1/β-Klotho-bindingprotein chimeras in SEQ ID NOS: 173, 174, 175, 177, 178, 179, 181, 182,183, 185, 186, and 187, and mutant FGF1/FGFR1c-binding protein chimerasin SEQ ID NOS: 188 and 189. One skilled in the art will recognize thatminor variations can be made to these sequences, without adverselyaffecting the function of the protein (such as its ability to reduceblood glucose). For example, variants of the mutant FGF1 proteinsinclude those having at least 90%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 6, 7,8, 9, 10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106,107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,173, 174, 175, 177, 178, 179, 181, 182, 183, 185, 186, 187, 188, 189,191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204,205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218,219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232,233, 234, 235, 236, 237 or 238 (but are not a native FGF1 sequence,e.g., SEQ ID NO: 5), but retain the ability to treat a metabolicdisease, or decrease blood glucose in a mammal (such as a mammal withtype II diabetes). Thus, variants of SEQ ID NO: 6, 7, 8, 9, 10, 11, 12,13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 173, 174, 175, 177,178, 179, 181, 182, 183, 185, 186, 187, 188, 189, 191, 192, 193, 194,195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208,209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222,223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236,237 or 238 retaining at least 90%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% sequence identity are of use in thedisclosed methods.

FGF1

Mature forms of FGF1 (such as SEQ ID NO: 2 or 4) can be mutated tocontrol (e.g., reduce) the mitogenicity of the protein (for example bymutating the nuclear localization sequence (NLS) or the heparan sulfatebinding region or both) and to provide glucose-lowering ability to theprotein. Mutations can also be introduced into a wild-type mature FGF1sequence that affects the stability and receptor binding selectivity ofthe protein.

Exemplary full-length FGF1 proteins are shown in SEQ ID NOS: 2 (human)and 4 (mouse). In some examples, FGF1 includes SEQ ID NO: 2 or 4, butwithout the N-terminal methionine (thus resulting in a 154 aa FGF1protein). In addition, the mature/active form of FGF1 is one where aportion of the N-terminus is removed, such as the N-terminal 15, 16, 20,or 21 amino acids from SEQ ID NO: 2 or 4. Thus, in some examples theactive form of FGF1 comprises or consists of amino acids 16-155 or22-155 of SEQ ID NO: 2 or 4 (e.g., see SEQ ID NO: 5). In some examples,the mature form of FGF1 that can be mutated includes SEQ ID NO: 5 with amethionine added to the N-terminus (wherein such a sequence can bemutated as discussed herein). Thus, the mutated mature FGF1 protein caninclude an N-terminal truncation.

Mutations can be introduced into a wild-type FGF1 (such as SEQ ID NO: 2,4, or 5). In some examples, multiple types of mutations disclosed hereinare made to the FGF1 protein. Although mutations below are noted by aparticular amino acid for example in SEQ ID NO: 2, 4 or 5, one skilledin the art will appreciate that the corresponding amino acid can bemutated in any FGF1 sequence. For example, Q40 of SEQ ID NO: 5corresponds to Q55 of SEQ ID NO: 2 and 4.

In one example, mutations are made to the N-terminal region of FGF1(such as SEQ ID NO: 2, 4 or 5), such as deletion of the first 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 aminoacids of SEQ ID NO: 2 or 4 (such as deletion of at least the first 14amino acids of SEQ ID NO: 2 or 4, such as deletion of at least the first15, at least 16, at least 20, at least 25, or at least 29 amino acids ofSEQ ID NO: 2 or 4), deletion of the first 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or 20 amino acids of SEQ ID NO: 5 (e.g., see SEQ ID NOS:7, 8 and 9 and FIG. 1).

Mutations can be made to FGF1 (such as SEQ ID NO: 2, 4 or 5) to reduceits mitogenic activity. In some examples, such mutations reducemitogenic activity by at least 50%, at least 60%, at least 70%, at least75%, at least 80%, at least 90%, at least 92%, at least 95%, at least98%, at least 99%, or even complete elimination of detectable mitogenicactivity, as compared to a native FGF1 protein without the mutation.Methods of measuring mitogenic activity are known in the art, such asthymidine incorporation into DNA in serum-starved cells (e.g., NIH 3T3cells) stimulated with the mutated FGF1, methylthiazoletetrazolium (MTT)assay (for example by stimulating serum-starved cells with mutated FGF1for 24 hr then measuring viable cells), cell number quantification orBrdU incorporation. In some examples, the assay provided by Fu et al.,World J. Gastroenterol. 10:3590-6, 2004; Klingenberg et al., J. Biol.Chem. 274:18081-6, 1999; Shen et al., Protein Expr Purif. 81:119-25,2011, or Zou et al., Chin. Med. J. 121:424-429, 2008 is used to measuremitogenic activity. Examples of such mutations include, but are notlimited to K12V, R35E, L46V, E87V, N95V, K12V/N95V (e.g., see SEQ ID NO:10, which can also include a methionine on its N-terminus), andLys12Val/Pro134Val, Lys12Val/Leu46Val/Glu87Val/Asn95Val/Pro134Val (e.g.,see SEQ ID NO: 11, which can also include a methionine on itsN-terminus) (wherein the numbering refers to the sequence shown SEQ IDNO: 5). In some examples, a portion of contiguous N-terminal residuesare removed, such as amino acids 1-9 of SEQ ID NO: 5, to produce anon-mitogenic form of FGF1. An example is shown in SEQ ID NO: 9.

Mutations that reduce the heparan binding affinity (such as a reductionof at least 10%, at least 20%, at least 50%, or at least 75%, e.g., ascompared to a native FGF1 protein without the mutation), can also beused to reduce mitogenic activity, for example by substituting heparanbinding residues from a paracrine FGFs into FGF1. In some examples,mitogenicity is reduced or eliminated by deleting the N-terminal regionof FGF1 (such as the region that binds FGF4) and replacing some or allof the amino acids deleted with corresponding residues from FGF21.

Mutations can also be introduced into one or both nuclear localizationsites (NLS1, amino acids 24-27 of SEQ ID NO: 2 and NLS2, amino acids115-128 of SEQ ID NO: 4) of FGF1, for example to reduce mitogenicity, ascompared to a native FGF1 protein without the mutation. Examples of NLSmutations that can be made to FGF1 include but are not limited to:deleting or mutating all or a part of NLS1 (such as deleting or mutatingthe lysines), deleting or mutating the lysines in NLS2 such as ¹¹⁵KK . .. ¹²⁷KK . . . , or combinations thereof (wherein the numbering refers tothe sequence shown SEQ ID NO: 2). For example, one or more of 24K, 25K,27K, 115K, 127K or 128K (wherein the numbering refers to the sequenceshown SEQ ID NO: 2) or can be mutated (for example changed to an alanineor deleted). Particular examples of such mutations that can be made tothe heparan binding site in the NLS2 (KKN . . . KR) domain are shown inSEQ ID NOS: 12 and 13 (K118N or K118E, respectively, wherein numberingrefers to SEQ ID NO: 5).

Mutations can be introduced into the phosphorylation site of FGF1, forexample to create a constitutively active or inactive mutant to affectnuclear signaling.

In some examples, mutations are introduced into the FGF1 nuclear exportsequence, for example to decrease the amount of FGF 1 in the nucleus andreduce its mitogenicity as measured by thymidine incorporation assays incultured cells (e.g., see Nilsen et al., J. Biol. Chem.282(36):26245-56, 2007). Mutations to the nuclear export sequencedecrease FGF1-induced proliferation (e.g., see Nilsen et al., J. Biol.Chem. 282(36):26245-56, 2007). Methods of measuring FGF1 degradation areknown in the art, such as measuring [³⁵S]Methionine-labeled FGF1 orimmunoblotting for steady-state levels of FGF1 in the presence orabsence of proteasome inhibitors. In one example, the assay provided byNilsen et al., J. Biol. Chem. 282(36):26245-56, 2007 or Zakrzewska etal., J. Biol. Chem. 284:25388-403, 2009 is used to measure FGF1degradation.

The FGF1 nuclear export sequence includes amino acids 145-152 of SEQ IDNO: 2 and 4 or amino acids 130-137 of SEQ ID NO: 5. Examples of FGF1nuclear export sequence mutations that can be made to include but arenot limited to changing the sequence ILFLPLPV (amino acids 145-152 ofSEQ ID NO: 2 and 4) to AAALPLPV (SEQ ID NO: 14), ILALPLPV (SEQ ID NO:15), ILFAPLPV (SEQ ID NO: 16), or ILFLPAPA (SEQ ID NO: 17).

In one example, mutations are introduced to improve stability of FGF1.In some examples, the sequence NYKKPKL (amino acids 22-28 of SEQ ID NO:2) is not altered, and in some examples ensures for structural integrityof FGF1 and increases interaction with the FGF1 receptor. Methods ofmeasuring FGF1 stability are known in the art, such as measuringdenaturation of FGF1 or mutants by fluorescence and circular dichroismin the absence and presence of a 5-fold molar excess of heparin in thepresence of 1.5 M urea or isothermal equilibrium denaturation byguanidine hydrochloride. In one example, the assay provided by Dubey etal., J. Mol. Biol. 371:256-268, 2007 is used to measure FGF1 stability.Examples of mutations that can be used to increase stability of theprotein include, but are not limited to, one or more of Q40P, S47I andH93G (wherein the numbering refers to the sequence shown SEQ ID NO: 5).

In one example, mutations are introduced to improve the thermostabilityof FGF1, such as an increase of at least 10%, at least 20%, at least50%, or at least 75%, as compared to a native FGF1 protein without themutation (e.g., see Xia et al., PLoS One. 2012; 7(11):e48210 andZakrzewska, J Biol. Chem. 284:25388-25403, 2009). In one example,mutations are introduced to increase protease resistance of FGF1 (e.g.,see Kobielak et al., Protein Pept Lett. 21(5):434-43, 2014). Othermutations that can be made to FGF1 include those mutations provided inLin et al., J Biol. Chem. 271(10):5305-8, 1996).

In some examples, the mutant FGF1 protein or chimera is PEGylated at oneor more positions, such as at N95 (for example see methods of Niu etal., J. Chromatog. 1327:66-72, 2014, herein incorporated by reference).Pegylation consists of covalently linking a polyethylene glycol group tosurface residues and/or the N-terminal amino group. N95 is known to beinvolved in receptor binding, thus is on the surface of the foldedprotein. As mutations to surface exposed residues could potentiallygenerate immunogenic sequences, pegylation is an alternative method toabrogate a specific interaction. Pegylation is an option for any surfaceexposed site implicated in the receptor binding and/or proteolyticdegradation. Pegylation can “cover” functional amino acids, e.g. N95, aswell as increase serum stability.

In some examples, the mutant FGF1 protein or chimera includes animmunoglobin FC domain (for example see Czajkowsky et al., EMBO Mol.Med. 4:1015-28, 2012, herein incorporated by reference). The conservedFC fragment of an antibody can be incorporated either n-terminal orc-terminal of the mutant FGF1 protein or chimera, and can enhancestability of the protein and therefore serum half-life. The FC domaincan also be used as a means to purify the proteins on protein A orProtein G sepharose beads. This makes the FGF1 mutants having heparinbinding mutations easier to purify.

Variant Sequences

Variant FGF1 proteins, including variants of the sequences shown inTables 1 and 2, and variants of SEQ ID NOS: 6, 7, 8, 9, 10, 11, 12, 13,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,112, 113, 114, 115, 116, 117, 118, 119, 120, 173, 174, 175, 177, 178,179, 181, 182, 183, 185, 186, 187, 188, 189, 191, 192, 193, 194, 195,196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209,210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223,224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237,and 238, can contain one or more mutations, such as a single insertion,a single deletion, a single substitution. In some examples, the mutantFGF1 protein includes 1-20 insertions, 1-20 deletions, 1-20substitutions, or any combination thereof (e.g., single insertiontogether with 1-19 substitutions). In some examples, the disclosureprovides a variant of any disclosed mutant FGF1 protein having 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 aminoacid changes. In some examples, SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,112, 113, 114, 115, 116, 117, 118, 119, 120, 173, 174, 175, 177, 178,179, 181, 182, 183, 185, 186, 187, 188, 189, 191, 192, 193, 194, 195,196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209,210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223,224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or238, includes 1-8 insertions, 1-15 deletions, 1-10 substitutions, or anycombination thereof (e.g., 1-15, 1-4, or 1-5 amino acid deletionstogether with 1-10, 1-5 or 1-7 amino acid substitutions). In someexamples, the disclosure provides a variant of any of SEQ ID NOS: 6, 7,8, 9, 10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106,107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,173, 174, 175, 177, 178, 179, 181, 182, 183, 185, 186, 187, 188, 189,191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204,205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218,219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232,233, 234, 235, 236, 237 and 238, having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29 or 30 amino acid changes. In one example, such variant peptides areproduced by manipulating the nucleotide sequence encoding a peptideusing standard procedures such as site-directed mutagenesis or PCR. Suchvariants can also be chemically synthesized. Similar changes can be madeto the FGFR1c dimer of SEQ ID NO: 190 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid changes).

One type of modification or mutation includes the substitution of aminoacids for amino acid residues having a similar biochemical property,that is, a conservative substitution (such as 1-4, 1-8, 1-10, or 1-20conservative substitutions). Typically, conservative substitutions havelittle to no impact on the activity of a resulting peptide. For example,a conservative substitution is an amino acid substitution in SEQ ID NO:6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105,106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 173, 174, 175, 177, 178, 179, 181, 182, 183, 185, 186, 187, 188,189, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203,204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217,218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231,232, 233, 234, 235, 236, 237 or 238, that does not substantially affectthe ability of the peptide to decrease blood glucose in a mammal. Analanine scan can be used to identify which amino acid residues in amutant FGF1 protein, such as SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 173, 174, 175, 177, 178, 179,181, 182, 183, 185, 186, 187, 188, 189, 191, 192, 193, 194, 195, 196,197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210,211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224,225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238,can tolerate an amino acid substitution. In one example, the bloodglucose lowering activity of FGF1, or any of SEQ ID NO: 6, 7, 8, 9, 10,11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 173, 174, 175,177, 178, 179, 181, 182, 183, 185, 186, 187, 188, 189, 191, 192, 193,194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207,208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235,236, 237 or 238, is not altered by more than 25%, for example not morethan 20%, for example not more than 10%, when an alanine, or otherconservative amino acid, is substituted for 1-4, 1-8, 1-10, or 1-20native amino acids. Examples of amino acids which may be substituted foran original amino acid in a protein and which are regarded asconservative substitutions include: Ser for Ala; Lys for Arg; Gln or Hisfor Asn; Glu for Asp; Ser for Cys; Asn for Gln; Asp for Glu; Pro forGly; Asn or Gln for His; Leu or Val for Ile; Ile or Val for Leu; Arg orGln for Lys; Leu or Ile for Met; Met, Leu or Tyr for Phe; Thr for Ser;Ser for Thr; Tyr for Trp; Trp or Phe for Tyr; and Ile or Leu for Val.

More substantial changes can be made by using substitutions that areless conservative, e.g., selecting residues that differ moresignificantly in their effect on maintaining: (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation; (b) the charge or hydrophobicity of thepolypeptide at the target site; or (c) the bulk of the side chain. Thesubstitutions that in general are expected to produce the greatestchanges in polypeptide function are those in which: (a) a hydrophilicresidue, e.g., serine or threonine, is substituted for (or by) ahydrophobic residue, e.g., leucine, isoleucine, phenylalanine, valine oralanine; (b) a cysteine or proline is substituted for (or by) any otherresidue; (c) a residue having an electropositive side chain, e.g.,lysine, arginine, or histidine, is substituted for (or by) anelectronegative residue, e.g., glutamic acid or aspartic acid; or (d) aresidue having a bulky side chain, e.g., phenylalanine, is substitutedfor (or by) one not having a side chain, e.g., glycine. The effects ofthese amino acid substitutions (or other deletions or additions) can beassessed by analyzing the function of the mutant FGF1 protein, such asany of SEQ ID NOS: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 173, 174, 175, 177, 178, 179, 181, 182, 183,185, 186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197, 198, 199,200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227,228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238, by analyzingthe ability of the variant protein to decrease blood glucose in amammal.

Generation of Proteins

Isolation and purification of recombinantly expressed mutated FGF1proteins can be carried out by conventional means, such as preparativechromatography and immunological separations. Once expressed, mutatedFGF1 proteins can be purified according to standard procedures of theart, including ammonium sulfate precipitation, affinity columns, columnchromatography, and the like (see, generally, R. Scopes, ProteinPurification, Springer-Verlag, N.Y., 1982). Substantially purecompositions of at least about 90 to 95% homogeneity are disclosedherein, and 98 to 99% or more homogeneity can be used for pharmaceuticalpurposes.

In addition to recombinant methods, mutated FGF1 proteins disclosedherein can also be constructed in whole or in part using standardpeptide synthesis. In one example, mutated FGF1 proteins are synthesizedby condensation of the amino and carboxyl termini of shorter fragments.Methods of forming peptide bonds by activation of a carboxyl terminalend (such as by the use of the coupling reagentN,N′-dicylohexylcarbodimide) are well known in the art.

Mutated FGF1 and FGFR1c-Binding Protein Multimer Nucleic Acid Moleculesand Vectors

Nucleic acid molecules encoding a mutated FGF1 protein are encompassedby this disclosure. Based on the genetic code, nucleic acid sequencescoding for any mutated FGF1 sequence, such as those generated using thesequences shown in Tables 1 and 2, can be routinely generated.Similarly, mutant FGF1/β-Klotho-binding, mutant FGF1/FGFR1c-binding,mutant FGF1/β-Klotho-binding/FGFR1c-binding, mutant FGF1/FGF21 or mutantFGF1/FGF19 chimeras can be generated using routine methods based on theamino acid sequences provided herein. In some examples, such a sequenceis optimized for expression in a host cell, such as a host cell used toexpress the mutant FGF1 protein. Also provided are nucleic acidmolecules encoding an FGFR1c-binding protein multimer, such as thoseencoding multimers of any of SEQ ID NOS: 147, 148, 149, 150, 151, 152,153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166,167, or 190, as well as cells and vectors including such nucleic acids.

In one example, a nucleic acid sequence codes for a mutant FGF1 protein(or chimera including such protein) having at least 60%, at least 70%,at least 75%, at least 80%, at least 90%, at least 92%, at least 95%, atlest 96%, at least 97%, at least 99% or at least 99% sequence identityto SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101, 102,103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116,117, 118, 119, 120, 173, 174, 175, 177, 178, 179, 181, 182, 183, 185,186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200,201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214,215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228,229, 230, 231, 232, 233, 234, 235, 236, 237 or 238, can readily beproduced by one of skill in the art, using the amino acid sequencesprovided herein, and the genetic code. In addition, one of skill canreadily construct a variety of clones containing functionally equivalentnucleic acids, such as nucleic acids which differ in sequence but whichencode the same mutant FGF1 protein sequence. In one example, a mutantFGF1 nucleic acid sequence has at least 70%, at least 80%, at least 85%,at least 90%, at least 92%, at least 95%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 18.

In one example, a nucleic acid sequence codes for a FGFR1c-bindingprotein multimer made using peptide sequences having at least 60%, atleast 70%, at least 75%, at least 80%, at least 90%, at least 92%, atleast 95%, at lest 96%, at least 97%, at least 99% or at least 99%sequence identity to SEQ ID NO: 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, or 190.

Nucleic acid molecules include DNA, cDNA and RNA sequences which encodea mutated FGF1 peptide. Silent mutations in the coding sequence resultfrom the degeneracy (i.e., redundancy) of the genetic code, whereby morethan one codon can encode the same amino acid residue. Thus, forexample, leucine can be encoded by CTT, CTC, CTA, CTG, TTA, or TTG;serine can be encoded by TCT, TCC, TCA, TCG, AGT, or AGC; asparagine canbe encoded by AAT or AAC; aspartic acid can be encoded by GAT or GAC;cysteine can be encoded by TGT or TGC; alanine can be encoded by GCT,GCC, GCA, or GCG; glutamine can be encoded by CAA or CAG; tyrosine canbe encoded by TAT or TAC; and isoleucine can be encoded by ATT, ATC, orATA. Tables showing the standard genetic code can be found in varioussources (see, for example, Stryer, 1988, Biochemistry, 3^(rd) Edition,W.H. 5 Freeman and Co., NY).

Codon preferences and codon usage tables for a particular species can beused to engineer isolated nucleic acid molecules encoding aFGFR1c-binding protein multimer or a mutated FGF1 protein (such as oneencoding a protein generated using the sequences shown in Tables 1 and2, the sequences in any of SEQ ID NOS: 21-84, 113-120 and 191-238 orthose encoding a protein having at least 90%, at least 95%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 173, 174, 175, 177, 178, 179, 181,182, 183, 185, 186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197,198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211,212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238) thattake advantage of the codon usage preferences of that particularspecies. For example, the FGFR1c-binding protein multimers and mutatedFGF1 proteins disclosed herein can be designed to have codons that arepreferentially used by a particular organism of interest.

A nucleic acid encoding a FGFR1c-binding protein multimer or a mutantFGF1 protein (such as one encoding a protein generated using thesequences shown in Tables 1 and 2, the sequences in any of SEQ ID NOS:21-84, 113-120 and 191-238 or those encoding a protein having at least90%, at least 95%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or 100% sequence identity to SEQ ID NO: 6, 7, 8, 9,10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 173, 174,175, 177, 178, 179, 181, 182, 183, 185, 186, 187, 188, 189, 191, 192,193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220,221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,235, 236, 237 or 238) can be cloned or amplified by in vitro methods,such as the polymerase chain reaction (PCR), the ligase chain reaction(LCR), the transcription-based amplification system (TAS), theself-sustained sequence replication system (3SR) and the Q13 replicaseamplification system (QB). A wide variety of cloning and in vitroamplification methodologies are well known to persons skilled in theart. In addition, nucleic acids encoding sequences encoding aFGFR1c-binding protein multimer or a mutant FGF1 protein (such as oneencoding a protein generated using the sequences shown in Tables 1 and2, the sequences in any of SEQ ID NOS: 21-84, 113-120 and 191-238 orthose encoding a protein having at least 90%, at least 95%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 173, 174, 175, 177, 178, 179, 181,182, 183, 185, 186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197,198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211,212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238) canbe prepared by cloning techniques. Examples of appropriate cloning andsequencing techniques, and instructions sufficient to direct persons ofskill through cloning are found in Sambrook et al. (ed.), MolecularCloning: A Laboratory Manual 2nd ed., vol. 1-3, Cold Spring HarborLaboratory Press, Cold Spring, Harbor, N.Y., 1989, and Ausubel et al.,(1987) in “Current Protocols in Molecular Biology,” John Wiley and Sons,New York, N.Y.

Nucleic acid sequences encoding a FGFR1c-binding protein multimer or amutated FGF1 protein (such as one encoding a protein generated using thesequences shown in Tables 1 and 2, the sequences in any of SEQ ID NOS:21-84, 113-120 and 191-238 or those encoding a protein having at least90%, at least 95%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or 100% sequence identity to SEQ ID NO: 6, 7, 8, 9,10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 173, 174,175, 177, 178, 179, 181, 182, 183, 185, 186, 187, 188, 189, 191, 192,193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220,221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,235, 236, 237 or 238) can be prepared by any suitable method including,for example, cloning of appropriate sequences or by direct chemicalsynthesis by methods such as the phosphotriester method of Narang etal., Meth. Enzymol. 68:90-99, 1979; the phosphodiester method of Brownet al., Meth. Enzymol. 68:109-151, 1979; the diethylphosphoramiditemethod of Beaucage et al., Tetra. Lett. 22:1859-1862, 1981; the solidphase phosphoramidite triester method described by Beaucage & Caruthers,Tetra. Letts. 22(20):1859-1862, 1981, for example, using an automatedsynthesizer as described in, for example, Needham-VanDevanter et al.,Nucl. Acids Res. 12:6159-6168, 1984; and, the solid support method ofU.S. Pat. No. 4,458,066. Chemical synthesis produces a single strandedoligonucleotide. This can be converted into double stranded DNA byhybridization with a complementary sequence, or by polymerization with aDNA polymerase using the single strand as a template. One of skill wouldrecognize that while chemical synthesis of DNA is generally limited tosequences of about 100 bases, longer sequences may be obtained by theligation of shorter sequences.

In one example, a mutant FGF1 protein (such as a protein generated usingthe sequences shown in Tables 1 and 2, the sequences in any of SEQ IDNOS: 21-84, 113-120 and 191-238 or those encoding a protein having atleast 90%, at least 95%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or 100% sequence identity to SEQ ID NO: 6, 7, 8,9, 10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107,108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 173,174, 175, 177, 178, 179, 181, 182, 183, 185, 186, 187, 188, 189, 191,192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205,206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219,220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233,234, 235, 236, 237 or 238) is prepared by inserting the cDNA whichencodes the mutant FGF1 protein into a vector. The insertion can be madeso that the mutant FGF1 protein is read in frame so that the mutant FGF1protein is produced. Similar methods can be used for a FGFR1c-bindingprotein multimer.

The mutated FGF1 protein nucleic acid coding sequence (such as oneencoding a protein generated using the sequences shown in Tables 1 and2, the sequences in any of SEQ ID NOS: 21-84, 113-120 and 191-238 orthose encoding a protein having at least 90%, at least 95%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 173, 174, 175, 177, 178, 179, 181,182, 183, 185, 186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197,198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211,212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238) canbe inserted into an expression vector including, but not limited to aplasmid, virus or other vehicle that can be manipulated to allowinsertion or incorporation of sequences and can be expressed in eitherprokaryotes or eukaryotes. Hosts can include microbial, yeast, insect,plant and mammalian organisms. Methods of expressing DNA sequenceshaving eukaryotic or viral sequences in prokaryotes are well known inthe art. Biologically functional viral and plasmid DNA vectors capableof expression and replication in a host are known in the art. The vectorcan encode a selectable marker, such as a thymidine kinase gene. Similarmethods can be used for a FGFR1c-binding protein multimer.

Nucleic acid sequences encoding a FGFR1c-binding protein multimer or amutated FGF1 protein (such as one encoding a protein generated using thesequences shown in Tables 1 and 2, the sequences in any of SEQ ID NOS:21-84, 113-120 and 191-238 or those encoding a protein having at least90%, at least 95%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or 100% sequence identity to SEQ ID NO: 6, 7, 8, 9,10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 173, 174,175, 177, 178, 179, 181, 182, 183, 185, 186, 187, 188, 189, 191, 192,193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220,221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,235, 236, 237 or 238) can be operatively linked to expression controlsequences. An expression control sequence operatively linked to aFGFR1c-binding protein multimer or mutated FGF1 protein coding sequenceis ligated such that expression of the FGFR1c-binding protein multimeror mutant FGF1 protein coding sequence is achieved under conditionscompatible with the expression control sequences. The expression controlsequences include, but are not limited to appropriate promoters,enhancers, transcription terminators, a start codon (i.e., ATG) in frontof a FGFR1c-binding protein multimer or mutated FGF1 protein-encodinggene, splicing signal for introns, maintenance of the correct readingframe of that gene to permit proper translation of mRNA, and stopcodons.

In one embodiment, vectors are used for expression in yeast such as S.cerevisiae, P. pastoris, or Kluyveromyces lactis. Several promoters areknown to be of use in yeast expression systems such as the constitutivepromoters plasma membrane H⁺-ATPase (PMA1), glyceraldehyde-3-phosphatedehydrogenase (GPD), phosphoglycerate kinase-1 (PGK1), alcoholdehydrogenase-1 (ADH1), and pleiotropic drug-resistant pump (PDR5). Inaddition, many inducible promoters are of use, such as GAL1-10 (inducedby galactose), PHO5 (induced by low extracellular inorganic phosphate),and tandem heat shock HSE elements (induced by temperature elevation to37° C.). Promoters that direct variable expression in response to atitratable inducer include the methionine-responsive MET3 and MET25promoters and copper-dependent CUP1 promoters. Any of these promotersmay be cloned into multicopy (2μ) or single copy (CEN) plasmids to givean additional level of control in expression level. The plasmids caninclude nutritional markers (such as URA3, ADE3, HIS1, and others) forselection in yeast and antibiotic resistance (AMP) for propagation inbacteria. Plasmids for expression on K. lactis are known, such aspKLAC1. Thus, in one example, after amplification in bacteria, plasmidscan be introduced into the corresponding yeast auxotrophs by methodssimilar to bacterial transformation. The nucleic acid molecules encodinga FGFR1c-binding protein multimer or a mutated FGF1 protein (such as oneencoding a protein generated using the sequences shown in Tables 1 and2, the sequences in any of SEQ ID NOS: 21-84, 113-120 and 191-238 orthose encoding a protein having at least 90%, at least 95%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 173, 174, 175, 177, 178, 179, 181,182, 183, 185, 186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197,198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211,212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238) canalso be designed to express in insect cells.

A FGFR1c-binding protein multimer or mutated FGF1 protein (such as aprotein generated using the sequences shown in Tables 1 and 2, thesequences in any of SEQ ID NOS: 21-84, 113-120 and 191-238 or thoseencoding a protein having at least 90%, at least 95%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or 100% sequenceidentity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 173, 174, 175, 177, 178, 179, 181, 182, 183,185, 186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197, 198, 199,200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227,228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238) can beexpressed in a variety of yeast strains. For example, seven pleiotropicdrug-resistant transporters, YOR1, SNQ2, PDR5, YCF1, PDR10, PDR11, andPDR15, together with their activating transcription factors, PDR1 andPDR3, have been simultaneously deleted in yeast host cells, renderingthe resultant strain sensitive to drugs. Yeast strains with alteredlipid composition of the plasma membrane, such as the erg6 mutantdefective in ergosterol biosynthesis, can also be utilized. Proteinsthat are highly sensitive to proteolysis can be expressed in a yeastcell lacking the master vacuolar endopeptidase Pep4, which controls theactivation of other vacuolar hydrolases. Heterologous expression instrains carrying temperature-sensitive (ts) alleles of genes can beemployed if the corresponding null mutant is inviable.

Viral vectors can also be prepared that encode a FGFR1c-binding proteinmultimer or a mutated FGF1 protein (such as one encoding a proteingenerated using the sequences shown in Tables 1 and 2, the sequences inany of SEQ ID NOS: 21-84, 113-120 and 191-238 or those encoding aprotein having at least 90%, at least 95%, at least 95%, at least 96%,at least 97%, at least 98%, at least 99% or 100% sequence identity toSEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103,104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,118, 119, 120, 173, 174, 175, 177, 178, 179, 181, 182, 183, 185, 186,187, 188, 189, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201,202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215,216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229,230, 231, 232, 233, 234, 235, 236, 237 or 238). Exemplary viral vectorsinclude polyoma, SV40, adenovirus, vaccinia virus, adeno-associatedvirus, herpes viruses including HSV and EBV, Sindbis viruses,alphaviruses and retroviruses of avian, murine, and human origin.Baculovirus (Autographa californica multinuclear polyhedrosis virus;AcMNPV) vectors are also known in the art, and may be obtained fromcommercial sources. Other suitable vectors include retrovirus vectors,orthopox vectors, avipox vectors, fowlpox vectors, capripox vectors,suipox vectors, adenoviral vectors, herpes virus vectors, alpha virusvectors, baculovirus vectors, Sindbis virus vectors, vaccinia virusvectors and poliovirus vectors. Specific exemplary vectors are poxvirusvectors such as vaccinia virus, fowlpox virus and a highly attenuatedvaccinia virus (MVA), adenovirus, baculovirus and the like. Pox virusesof use include orthopox, suipox, avipox, and capripox virus. Orthopoxinclude vaccinia, ectromelia, and raccoon pox. One example of anorthopox of use is vaccinia. Avipox includes fowlpox, canary pox andpigeon pox. Capripox include goatpox and sheeppox. In one example, thesuipox is swinepox. Other viral vectors that can be used include otherDNA viruses such as herpes virus and adenoviruses, and RNA viruses suchas retroviruses and polio.

Viral vectors that encode a FGFR1c-binding protein multimer or a mutatedFGF1 protein (such as one encoding a protein generated using thesequences shown in Tables 1 and 2, the sequences in any of SEQ ID NOS:21-84, 113-120 and 191-238 or those encoding a protein having at least90%, at least 95%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or 100% sequence identity to SEQ ID NO: 6, 7, 8, 9,10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 173, 174,175, 177, 178, 179, 181, 182, 183, 185, 186, 187, 188, 189, 191, 192,193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220,221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,235, 236, 237 or 238) can include at least one expression controlelement operationally linked to the nucleic acid sequence encoding theFGFR1c-binding protein multimer or mutated FGF1 protein. The expressioncontrol elements are inserted in the vector to control and regulate theexpression of the nucleic acid sequence. Examples of expression controlelements of use in these vectors includes, but is not limited to, lacsystem, operator and promoter regions of phage lambda, yeast promotersand promoters derived from polyoma, adenovirus, retrovirus or SV40.Additional operational elements include, but are not limited to, leadersequence, termination codons, polyadenylation signals and any othersequences necessary for the appropriate transcription and subsequenttranslation of the nucleic acid sequence encoding the mutated FGF1protein in the host system. The expression vector can contain additionalelements necessary for the transfer and subsequent replication of theexpression vector containing the nucleic acid sequence in the hostsystem. Examples of such elements include, but are not limited to,origins of replication and selectable markers. It will further beunderstood by one skilled in the art that such vectors are easilyconstructed using conventional methods (Ausubel et al., (1987) in“Current Protocols in Molecular Biology,” John Wiley and Sons, New York,N.Y.) and are commercially available.

Basic techniques for preparing recombinant DNA viruses containing aheterologous DNA sequence encoding the FGFR1c-binding protein multimeror mutated FGF1 protein (such as one encoding a protein generated usingthe sequences shown in Tables 1 and 2, the sequences in any of SEQ IDNOS: 21-84, 113-120 and 191-238 or those encoding a protein having atleast 90%, at least 95%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or 100% sequence identity to SEQ ID NO: 6, 7, 8,9, 10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107,108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 173,174, 175, 177, 178, 179, 181, 182, 183, 185, 186, 187, 188, 189, 191,192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205,206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219,220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233,234, 235, 236, 237 or 238) are known. Such techniques involve, forexample, homologous recombination between the viral DNA sequencesflanking the DNA sequence in a donor plasmid and homologous sequencespresent in the parental virus. The vector can be constructed for exampleby steps known in the art, such as by using a unique restrictionendonuclease site that is naturally present or artificially inserted inthe parental viral vector to insert the heterologous DNA.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate coprecipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors can be used. Eukaryotic cells can also beco-transformed with polynucleotide sequences encoding a FGFR1c-bindingprotein multimer or a mutated FGF1 protein (such as one encoding aprotein generated using the sequences shown in Tables 1 and 2, thesequences in any of SEQ ID NOS: 21-84, 113-120 and 191-238 or thoseencoding a protein having at least 90%, at least 95%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or 100% sequenceidentity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 173, 174, 175, 177, 178, 179, 181, 182, 183,185, 186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197, 198, 199,200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227,228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238), and a secondforeign DNA molecule encoding a selectable phenotype, such as the herpessimplex thymidine kinase gene. Another method is to use a eukaryoticviral vector, such as simian virus 40 (SV40) or bovine papilloma virus,to transiently infect or transform eukaryotic cells and express theprotein (see for example, Eukaryotic Viral Vectors, Cold Spring HarborLaboratory, Gluzman ed., 1982). One of skill in the art can readily usean expression systems such as plasmids and vectors of use in producingmutated FGF1 proteins in cells including higher eukaryotic cells such asthe COS, CHO, HeLa and myeloma cell lines.

Cells Expressing Mutated FGF1 Proteins or FGFR1c-Binding ProteinMultimers

A nucleic acid molecule encoding a mutated FGF1 protein disclosed herein(or chimeric protein including a mutant FGF1), or an FGFR1c-bindingprotein multimer disclosed herein, can be used to transform cells andmake transformed cells. Thus, cells expressing a FGFR1c-binding proteinmultimer (such as a FGFR1c-binding protein multimer made using peptideshaving at least 60%, at least 70%, at least 75%, at least 80%, at least90%, at least 92%, at least 95%, at lest 96%, at least 97%, at least99%, at least 99%, or 100% sequence identity to SEQ ID NO: 147, 148,149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162,163, 164, 165, 166, 167, or 190) or a mutated FGF1 protein (such as aprotein generated using the sequences shown in Tables 1 and 2, thesequences in any of SEQ ID NOS: 21-84, 113-120 and 191-238 or thoseencoding a protein having at least 90%, at least 95%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or 100% sequenceidentity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 173, 174, 175, 177, 178, 179, 181, 182, 183,185, 186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197, 198, 199,200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227,228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238), are disclosed.Cells expressing a mutated FGF1 protein disclosed herein, or expressingan FGFR1c-binding protein multimer, can be eukaryotic or prokaryotic.Examples of such cells include, but are not limited to bacteria, archea,plant, fungal, yeast, insect, and mammalian cells, such asLactobacillus, Lactococcus, Bacillus (such as B. subtilis), Escherichia(such as E. coli), Clostridium, Saccharomyces or Pichia (such as S.cerevisiae or P. pastoris), Kluyveromyces lactis, Salmonellatyphimurium, SF9 cells, C129 cells, 293 cells, Neurospora, andimmortalized mammalian myeloid and lymphoid cell lines.

Cells expressing a mutated FGF1 protein or an FGFR1c-binding proteinmultimer are transformed or recombinant cells. Such cells can include atleast one exogenous nucleic acid molecule that encodes a mutated FGF1protein, for example one encoding a protein generated using thesequences shown in Tables 1 and 2, the sequences in any of SEQ ID NOS:21-84, 113-120 and 191-238 or those encoding a protein having at least90%, at least 95%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or 100% sequence identity to SEQ ID NO: 6, 7, 8, 9,10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 173, 174,175, 177, 178, 179, 181, 182, 183, 185, 186, 187, 188, 189, 191, 192,193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220,221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,235, 236, 237 or 238. Such cells can include at least one exogenousnucleic acid molecule that encodes an FGFR1c-binding protein multimer,such as one encoding a protein made using two or more peptides having atleast 90%, at least 95%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or 100% sequence identity to SEQ ID NO: 147,148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161,162, 163, 164, 165, 166, 167, or 190. It is understood that all progenymay not be identical to the parental cell since there may be mutationsthat occur during replication. Methods of stable transfer, meaning thatthe foreign DNA is continuously maintained in the host cell, are knownin the art.

Transformation of a host cell with recombinant DNA may be carried out byconventional techniques as are well known. Where the host isprokaryotic, such as E. coli, competent cells which are capable of DNAuptake can be prepared from cells harvested after exponential growthphase and subsequently treated by the CaCl₂ method using procedures wellknown in the art. Alternatively, MgCl₂ or RbCl can be used.Transformation can also be performed after forming a protoplast of thehost cell if desired, or by electroporation. Techniques for thepropagation of mammalian cells in culture are well-known (see, Jakobyand Pastan (eds), 1979, Cell Culture. Methods in Enzymology, volume 58,Academic Press, Inc., Harcourt Brace Jovanovich, N.Y.). Examples ofcommonly used mammalian host cell lines are VERO and HeLa cells, CHOcells, and WI38, BHK, and COS cell lines, although cell lines may beused, such as cells designed to provide higher expression desirableglycosylation patterns, or other features. Techniques for thetransformation of yeast cells, such as polyethylene glycoltransformation, protoplast transformation and gene guns are also knownin the art.

Pharmaceutical Compositions That Include Mutated FGF1 Molecules and/orFGFR1c-Binding Protein Multimers

Pharmaceutical compositions that include a mutated FGF1 protein (such asa protein generated using the sequences shown in Tables 1 and 2, thesequences in any of SEQ ID NOS: 21-84, 113-120 and 191-238 or thoseencoding a protein having at least 90%, at least 95%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or 100% sequenceidentity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 173, 174, 175, 177, 178, 179, 181, 182, 183,185, 186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197, 198, 199,200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227,228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238) or a nucleicacid encoding these proteins, can be formulated with an appropriatepharmaceutically acceptable carrier, depending upon the particular modeof administration chosen. Similarly, the disclosure providespharmaceutical compositions that include one or more FGFR1c-bindingprotein multimers, such as multimers of SEQ ID NO: 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,165, 166, or 167, such as SEQ ID NO: 190 (or sequences having at least90%, at least 95%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or 100% sequence identity to SEQ ID NO: 147, 148, 149,150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163,164, 165, 166, 167, or 190).

In some embodiments, the pharmaceutical composition consists essentiallyof an FGFR1c-binding protein multimer or a mutated FGF1 protein (such asa protein generated using the sequences shown in Table 1, the sequencesin any of SEQ ID NOS: 21-84, or a protein having at least 90%, at least95%, at least 95%, at least 96%, at least 97%, at least 98%, at least99% or 100% sequence identity to any of SEQ ID NOS: 6-13, 21-84, 87-98,101-112, 173-175, 177-179, 181-183, 185-189, and 191-238) (or a nucleicacid encoding such a protein) and a pharmaceutically acceptable carrier.In these embodiments, additional therapeutically effective agents arenot included in the compositions.

In other embodiments, the pharmaceutical composition includes anFGFR1c-binding protein multimer or a mutated FGF1 protein (such as aprotein generated using the sequences shown in Tables 1 and 2, thesequences in any of SEQ ID NOS: 21-84, 113-120 and 191-238 or thoseencoding a protein having at least 90%, at least 95%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or 100% sequenceidentity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 173, 174, 175, 177, 178, 179, 181, 182, 183,185, 186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197, 198, 199,200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227,228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238) (or a nucleicacid encoding such a protein) and a pharmaceutically acceptable carrier.Additional therapeutic agents, such as agents for the treatment ofdiabetes, can be included. Thus, the pharmaceutical compositions caninclude a therapeutically effective amount of another agent. Examples ofsuch agents include, without limitation, anti-apoptotic substances suchas the Nemo-Binding Domain and compounds that induce proliferation suchas cyclin dependent kinase (CDK)-6, CDK-4 and cyclin D1. Other activeagents can be utilized, such as antidiabetic agents for example,metformin, sulphonylureas (e.g., glibenclamide, tolbutamide,glimepiride), nateglinide, repaglinide, thiazolidinediones (e.g.,rosiglitazone, pioglitazone), peroxisome proliferator-activated receptor(PPAR)-gamma-agonists (such as C1262570, aleglitazar, farglitazar,muraglitazar, tesaglitazar, and TZD) and PPAR-γ antagonists,PPAR-gamma/alpha modulators (such as KRP 297), alpha-glucosidaseinhibitors (e.g., acarbose, voglibose), dipeptidyl peptidase (DPP)-IVinhibitors (such as LAF237, MK-431), alpha2-antagonists, agents forlowering blood sugar, cholesterol-absorption inhibitors,3-hydroxy-3-methylglutaryl-coenzyme A (HMGCoA) reductase inhibitors(such as a statin), insulin and insulin analogues, GLP-1 and GLP-1analogues (e.g. exendin-4) or amylin. Additional examples includeimmunomodulatory factors such as anti-CD3 mAb, growth factors such asHGF, VEGF, PDGF, lactogens, and PTHrP. In some examples, thepharmaceutical compositions containing a mutated FGF1 protein canfurther include a therapeutically effective amount of other FGFs, suchas FGF21, FGF19, or both, heparin, or combinations thereof.

The pharmaceutically acceptable carriers and excipients useful in thisdisclosure are conventional. See, e.g., Remington: The Science andPractice of Pharmacy, The University of the Sciences in Philadelphia,Editor, Lippincott, Williams, & Wilkins, Philadelphia, Pa., 21^(st)Edition (2005). For instance, parenteral formulations usually includeinjectable fluids that are pharmaceutically and physiologicallyacceptable fluid vehicles such as water, physiological saline, otherbalanced salt solutions, aqueous dextrose, glycerol or the like. Forsolid compositions (e.g., powder, pill, tablet, or capsule forms),conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, pH buffering agents, or the like, for example sodiumacetate or sorbitan monolaurate. Excipients that can be included are,for instance, other proteins, such as human serum albumin or plasmapreparations.

In some embodiments, an FGFR1c-binding protein multimer or a mutatedFGF1 protein (such as a protein generated using the sequences shown inTables 1 and 2, the sequences in any of SEQ ID NOS: 21-84, 113-120 and191-238 or those encoding a protein having at least 90%, at least 95%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 173, 174, 175, 177, 178, 179, 181,182, 183, 185, 186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197,198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211,212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238) isincluded in a controlled release formulation, for example, amicroencapsulated formulation. Various types of biodegradable andbiocompatible polymers, methods can be used, and methods ofencapsulating a variety of synthetic compounds, proteins and nucleicacids, have been well described in the art (see, for example, U.S.Patent Publication Nos. 2007/0148074; 2007/0092575; and 2006/0246139;U.S. Pat. Nos. 4,522,811; 5,753,234; and 7,081,489; PCT Publication No.WO/2006/052285; Benita, Microencapsulation: Methods and IndustrialApplications, 2^(nd) ed., CRC Press, 2006).

In other embodiments, an FGFR1c-binding protein multimer or a mutatedFGF1 protein (such as a protein generated using the sequences shown inTables 1 and 2, the sequences in any of SEQ ID NOS: 21-84, 113-120 and191-238 or those encoding a protein having at least 90%, at least 95%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 173, 174, 175, 177, 178, 179, 181,182, 183, 185, 186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197,198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211,212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238) isincluded in a nanodispersion system. Nanodispersion systems and methodsfor producing such nanodispersions are well known to one of skill in theart. See, e.g., U.S. Pat. No. 6,780,324; U.S. Pat. Publication No.2009/0175953. For example, a nanodispersion system includes abiologically active agent and a dispersing agent (such as a polymer,copolymer, or low molecular weight surfactant). Exemplary polymers orcopolymers include polyvinylpyrrolidone (PVP), poly(D,L-lactic acid)(PLA), poly(D,L-lactic-co-glycolic acid (PLGA), poly(ethylene glycol).Exemplary low molecular weight surfactants include sodium dodecylsulfate, hexadecyl pyridinium chloride, polysorbates, sorbitans,poly(oxyethylene) alkyl ethers, poly(oxyethylene) alkyl esters, andcombinations thereof. In one example, the nanodispersion system includesPVP and ODP or a variant thereof (such as 80/20 w/w). In some examples,the nanodispersion is prepared using the solvent evaporation method, seefor example, Kanaze et al., Drug Dev. Indus. Pharm. 36:292-301, 2010;Kanaze et al., J. Appl. Polymer Sci. 102:460-471, 2006. With regard tothe administration of nucleic acids, one approach to administration ofnucleic acids is direct treatment with plasmid DNA, such as with amammalian expression plasmid. As described above, the nucleotidesequence encoding an FGFR1c-binding protein multimer or a mutated FGF1protein (such as a protein generated using the sequences shown in Tables1 and 2, the sequences in any of SEQ ID NOS: 21-84, 113-120 and 191-238or those encoding a protein having at least 90%, at least 95%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 173, 174, 175, 177, 178, 179, 181,182, 183, 185, 186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197,198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211,212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238) canbe placed under the control of a promoter to increase expression of theprotein.

Many types of release delivery systems are available and known. Examplesinclude polymer based systems such as poly(lactide-glycolide),copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters,polyhydroxybutyric acid, and polyanhydrides. Microcapsules of theforegoing polymers containing drugs are described in, for example, U.S.Pat. No. 5,075,109. Delivery systems also include non-polymer systems,such as lipids including sterols such as cholesterol, cholesterol estersand fatty acids or neutral fats such as mono- di- and tri-glycerides;hydrogel release systems; silastic systems; peptide based systems; waxcoatings; compressed tablets using conventional binders and excipients;partially fused implants; and the like. Specific examples include, butare not limited to: (a) erosional systems in which an FGFR1c-bindingprotein multimer or a mutated FGF1 protein (such as a protein generatedusing the sequences shown in Tables 1 and 2, the sequences in any of SEQID NOS: 21-84, 113-120 and 191-238 or those encoding a protein having atleast 90%, at least 95%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or 100% sequence identity to SEQ ID NO: 6, 7, 8,9, 10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107,108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 173,174, 175, 177, 178, 179, 181, 182, 183, 185, 186, 187, 188, 189, 191,192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205,206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219,220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233,234, 235, 236, 237 or 238), or polynucleotide encoding this protein, iscontained in a form within a matrix such as those described in U.S. Pat.Nos. 4,452,775; 4,667,014; 4,748,034; 5,239,660; and 6,218,371 and (b)diffusional systems in which an active component permeates at acontrolled rate from a polymer such as described in U.S. Pat. Nos.3,832,253 and 3,854,480. In addition, pump-based hardware deliverysystems can be used, some of which are adapted for implantation.

Use of a long-term sustained release implant may be particularlysuitable for treatment of chronic conditions, such as diabetes.Long-term release, as used herein, means that the implant is constructedand arranged to deliver therapeutic levels of the active ingredient forat least 30 days, and preferably 60 days. Long-term sustained releaseimplants are well known to those of ordinary skill in the art andinclude some of the release systems described above. These systems havebeen described for use with nucleic acids (see U.S. Pat. No. 6,218,371).For use in vivo, nucleic acids and peptides are preferably relativelyresistant to degradation (such as via endo- and exo-nucleases). Thus,modifications of the disclosed mutated FGF1 proteins, such as theinclusion of a C-terminal amide, can be used.

The dosage form of the pharmaceutical composition can be determined bythe mode of administration chosen. For instance, in addition toinjectable fluids, topical, inhalation, oral and suppositoryformulations can be employed. Topical preparations can include eyedrops, ointments, sprays, patches and the like. Inhalation preparationscan be liquid (e.g., solutions or suspensions) and include mists, spraysand the like. Oral formulations can be liquid (e.g., syrups, solutionsor suspensions), or solid (e.g., powders, pills, tablets, or capsules).Suppository preparations can also be solid, gel, or in a suspensionform. For solid compositions, conventional non-toxic solid carriers caninclude pharmaceutical grades of mannitol, lactose, cellulose, starch,or magnesium stearate. Actual methods of preparing such dosage forms areknown, or will be apparent, to those skilled in the art.

The pharmaceutical compositions that include an FGFR1c-binding proteinmultimer or a mutated FGF1 protein (such as a protein generated usingthe sequences shown in Tables 1 and 2, the sequences in any of SEQ IDNOS: 21-84, 113-120 and 191-238 or those encoding a protein having atleast 90%, at least 95%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or 100% sequence identity to SEQ ID NO: 6, 7, 8,9, 10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107,108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 173,174, 175, 177, 178, 179, 181, 182, 183, 185, 186, 187, 188, 189, 191,192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205,206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219,220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233,234, 235, 236, 237 or 238) can be formulated in unit dosage form,suitable for individual administration of precise dosages. In onenon-limiting example, a unit dosage contains from about 1 mg to about 1g of an FGFR1c-binding protein multimer or a mutated FGF1 protein (suchas a protein generated using the sequences shown in Tables 1 and 2, thesequences in any of SEQ ID NOS: 21-84, 113-120 and 191-238 or thoseencoding a protein having at least 90%, at least 95%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or 100% sequenceidentity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 173, 174, 175, 177, 178, 179, 181, 182, 183,185, 186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197, 198, 199,200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227,228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238), such as about10 mg to about 100 mg, about 50 mg to about 500 mg, about 100 mg toabout 900 mg, about 250 mg to about 750 mg, or about 400 mg to about 600mg. In other examples, a therapeutically effective amount of anFGFR1c-binding protein multimer or a mutated FGF 1 protein (such as aprotein generated using the sequences shown in Tables 1 and 2, thesequences in any of SEQ ID NOS: 21-84, 113-120 and 191-238 or thoseencoding a protein having at least 90%, at least 95%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or 100% sequenceidentity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 173, 174, 175, 177, 178, 179, 181, 182, 183,185, 186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197, 198, 199,200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227,228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238) is about 0.01mg/kg to about 50 mg/kg, for example, about 0.5 mg/kg to about 25 mg/kgor about 1 mg/kg to about 10 mg/kg. In other examples, a therapeuticallyeffective amount of an FGFR1c-binding protein multimer or a mutated FGF1protein (such as a protein generated using the sequences shown in Tables1 and 2, the sequences in any of SEQ ID NOS: 21-84, 113-120 and 191-238or those encoding a protein having at least 90%, at least 95%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 173, 174, 175, 177, 178, 179, 181,182, 183, 185, 186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197,198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211,212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238) isabout 1 mg/kg to about 5 mg/kg, for example about 2 mg/kg. In aparticular example, a therapeutically effective amount of anFGFR1c-binding protein multimer or a mutated FGF1 protein (such as aprotein generated using the sequences shown in Tables 1 and 2, thesequences in any of SEQ ID NOS: 21-84, 113-120 and 191-238 or thoseencoding a protein having at least 90%, at least 95%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or 100% sequenceidentity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 173, 174, 175, 177, 178, 179, 181, 182, 183,185, 186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197, 198, 199,200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227,228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238) includes about1 mg/kg to about 10 mg/kg, such as about 2 mg/kg.

Treatment Using Mutated FGF1 or FGFR1c-Binding Protein Multimers

The disclosed FGFR1c-binding protein multimers (such as a protein madeusing two or more peptides having at least 90%, at least 95%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%sequence identity to SEQ ID NO: 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, or 190)and mutated FGF1 proteins and chimeras (such as a protein generatedusing the sequences shown in Tables 1 and 2, the sequences in any of SEQID NOS: 21-84, 113-120 and 191-238 or those encoding a protein having atleast 90%, at least 95%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or 100% sequence identity to SEQ ID NO: 6, 7, 8,9, 10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107,108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 173,174, 175, 177, 178, 179, 181, 182, 183, 185, 186, 187, 188, 189, 191,192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205,206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219,220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233,234, 235, 236, 237 or 238), or nucleic acids encoding such proteins, canbe administered to a subject, for example to treat a metabolic disease,for example by reducing fed and fasting blood glucose, improving insulinsensitivity and glucose tolerance, reducing systemic chronicinflammation, ameliorating hepatic steatosis in a mammal, reducing foodintake, or combinations thereof.

The compositions of this disclosure that include an FGFR1c-bindingprotein multimer or a mutated FGF1 protein (such as a protein generatedusing the sequences shown in Tables 1 and 2, the sequences in any of SEQID NOS: 21-84, 113-120 and 191-238 or those encoding a protein having atleast 90%, at least 95%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or 100% sequence identity to SEQ ID NO: 6, 7, 8,9, 10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107,108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 173,174, 175, 177, 178, 179, 181, 182, 183, 185, 186, 187, 188, 189, 191,192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205,206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219,220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233,234, 235, 236, 237 or 238) (or nucleic acids encoding these molecules)can be administered to humans or other animals by any means, includingorally, intravenously, intramuscularly, intraperitoneally, intranasally,intradermally, intrathecally, subcutaneously, via inhalation or viasuppository. In one non-limiting example, the composition isadministered via injection. In some examples, site-specificadministration of the composition can be used, for example byadministering an FGFR1c-binding protein multimer or a mutated FGF1protein (such as a protein generated using the sequences shown in Tables1 and 2, the sequences in any of SEQ ID NOS: 21-84, 113-120 and 191-238or those encoding a protein having at least 90%, at least 95%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 173, 174, 175, 177, 178, 179, 181,182, 183, 185, 186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197,198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211,212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238) (or anucleic acid encoding these molecules) to pancreas tissue (for exampleby using a pump, or by implantation of a slow release form at the siteof the pancreas). The particular mode of administration and the dosageregimen will be selected by the attending clinician, taking into accountthe particulars of the case (e.g. the subject, the disease, the diseasestate involved, the particular treatment, and whether the treatment isprophylactic). Treatment can involve daily or multi-daily or less thandaily (such as weekly or monthly etc.) doses over a period of a few daysto months, or even years. For example, a therapeutically effectiveamount of an FGFR1c-binding protein multimer or a mutated FGF1 protein(such as a protein generated using the sequences shown in Tables 1 and2, the sequences in any of SEQ ID NOS: 21-84, 113-120 and 191-238 orthose encoding a protein having at least 90%, at least 95%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 173, 174, 175, 177, 178, 179, 181,182, 183, 185, 186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197,198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211,212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238) canbe administered in a single dose, twice daily, weekly, or in severaldoses, for example daily, or during a course of treatment. In aparticular non-limiting example, treatment involves once daily dose ortwice daily dose.

The amount of an FGFR1c-binding protein multimer or mutated FGF1 protein(such as a protein generated using the sequences shown in Tables 1 and2, the sequences in any of SEQ ID NOS: 21-84, 113-120 and 191-238 orthose encoding a protein having at least 90%, at least 95%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 173, 174, 175, 177, 178, 179, 181,182, 183, 185, 186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197,198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211,212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238)administered can be dependent on the subject being treated, the severityof the affliction, and the manner of administration, and is best left tothe judgment of the prescribing clinician. Within these bounds, theformulation to be administered will contain a quantity of theFGFR1c-binding protein multimer or mutated FGF1 protein in amountseffective to achieve the desired effect in the subject being treated. Atherapeutically effective amount of an FGFR1c-binding protein multimeror mutated FGF1 protein (such as a protein generated using the sequencesshown in Tables 1 and 2, the sequences in any of SEQ ID NOS: 21-84,113-120 and 191-238 or those encoding a protein having at least 90%, atleast 95%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100% sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11,12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 173, 174, 175,177, 178, 179, 181, 182, 183, 185, 186, 187, 188, 189, 191, 192, 193,194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207,208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235,236, 237 or 238) can be the amount of the mutant FGF1protein orFGFR1c-binding protein multimer, or a nucleic acid encoding thesemolecules that is necessary to treat diabetes or reduce blood glucoselevels (for example a reduction of at least 5%, at least 10% or at least20%, for example relative to no administration of the mutant FGF1 orFGFR1c-binding protein multimer).

When a viral vector is utilized for administration of an nucleic acidencoding an FGFR1c-binding protein multimer or a mutated FGF1 protein(such as a protein generated using the sequences shown in Tables 1 and2, the sequences in any of SEQ ID NOS: 21-84, 113-120 and 191-238 orthose encoding a protein having at least 90%, at least 95%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 173, 174, 175, 177, 178, 179, 181,182, 183, 185, 186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197,198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211,212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238), therecipient can receive a dosage of each recombinant virus in thecomposition in the range of from about 10⁵ to about 10¹⁰ plaque formingunits/mg mammal, although a lower or higher dose can be administered.Examples of methods for administering the composition into mammalsinclude, but are not limited to, exposure of cells to the recombinantvirus ex vivo, or injection of the composition into the affected tissueor intravenous, subcutaneous, intradermal or intramuscularadministration of the virus. Alternatively the recombinant viral vectoror combination of recombinant viral vectors may be administered locallyby direct injection into the pancreases in a pharmaceutically acceptablecarrier.

Generally, the quantity of recombinant viral vector, carrying thenucleic acid sequence of an FGFR1c-binding protein multimer or themutated FGF1 protein to be administered (such as a protein generatedusing the sequences shown in Tables 1 and 2, the sequences in any of SEQID NOS: 21-84, 113-120 and 191-238 or those encoding a protein having atleast 90%, at least 95%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or 100% sequence identity to SEQ ID NO: 6, 7, 8,9, 10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107,108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 173,174, 175, 177, 178, 179, 181, 182, 183, 185, 186, 187, 188, 189, 191,192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205,206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219,220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233,234, 235, 236, 237 or 238) is based on the titer of virus particles. Anexemplary range to be administered is 10⁵ to 10¹⁰ virus particles permammal, such as a human.

In some examples, an FGFR1c-binding protein multimer or mutated FGF1protein (such as a protein generated using the sequences shown in Tables1 and 2, the sequences in any of SEQ ID NOS: 21-84, 113-120 and 191-238or those encoding a protein having at least 90%, at least 95%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 173, 174, 175, 177, 178, 179, 181,182, 183, 185, 186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197,198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211,212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238), or anucleic acid encoding the FGFR1c-binding protein multimer or the mutatedFGF1 protein, is administered in combination (such as sequentially orsimultaneously or contemporaneously) with one or more other agents, suchas those useful in the treatment of diabetes or insulin resistance.

Anti-diabetic agents are generally categorized into six classes:biguanides (e.g., metformin); thiazolidinediones (includingrosiglitazone (Avandia®), pioglitazone (Actos®), rivoglitazone, andtroglitazone); sulfonylureas; inhibitors of carbohydrate absorption;fatty acid oxidase inhibitors and anti-lipolytic drugs; and weight-lossagents. Any of these agents can also be used in the methods disclosedherein. The anti-diabetic agents include those agents disclosed inDiabetes Care, 22(4):623-634. One class of anti-diabetic agents of useis the sulfonylureas, which are believed to increase secretion ofinsulin, decrease hepatic glucogenesis, and increase insulin receptorsensitivity. Another class of anti-diabetic agents use the biguanideantihyperglycemics, which decrease hepatic glucose production andintestinal absorption, and increase peripheral glucose uptake andutilization, without inducing hyperinsulinemia.

In some examples, an FGFR1c-binding protein multimer or mutated FGF1protein (such as a protein generated using the sequences shown in Tables1 and 2, the sequences in any of SEQ ID NOS: 21-84, 113-120 and 191-238or those encoding a protein having at least 90%, at least 95%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 173, 174, 175, 177, 178, 179, 181,182, 183, 185, 186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197,198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211,212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238) canbe administered in combination with effective doses of anti-diabeticagents (such as biguanides, thiazolidinediones, or incretins) and/orlipid lowering compounds (such as statins or fibrates). The term“administration in combination” or “co-administration” refers to bothconcurrent and sequential administration of the active agents.Administration of an FGFR1c-binding protein multimer or mutated FGF1protein (such as a protein generated using the sequences shown in Tables1 and 2, the sequences in any of SEQ ID NOS: 21-84, 113-120 and 191-238or those encoding a protein having at least 90%, at least 95%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 173, 174, 175, 177, 178, 179, 181,182, 183, 185, 186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197,198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211,212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 2388) or anucleic acid encoding such an FGFR1c-binding protein multimer or amutant FGF1 protein, may also be in combination with lifestylemodifications, such as increased physical activity, low fat diet, lowsugar diet, and smoking cessation.

Additional agents that can be used in combination with the disclosedFGFR1c-binding protein multimers and mutated FGF1 proteins include,without limitation, anti-apoptotic substances such as the Nemo-BindingDomain and compounds that induce proliferation such as cyclin dependentkinase (CDK)-6, CDK-4 and Cyclin D1. Other active agents can beutilized, such as antidiabetic agents for example, metformin,sulphonylureas (e.g., glibenclamide, tolbutamide, glimepiride),nateglinide, repaglinide, thiazolidinediones (e.g., rosiglitazone,pioglitazone), peroxisome proliferator-activated receptor(PPAR)-gamma-agonists (such as C1262570) and antagonists,PPAR-gamma/alpha modulators (such as KRP 297), alpha-glucosidaseinhibitors (e.g., acarbose, voglibose), Dipeptidyl peptidase (DPP)-IVinhibitors (such as LAF237, MK-431), alpha2-antagonists, agents forlowering blood sugar, cholesterol-absorption inhibitors,3-hydroxy-3-methylglutaryl-coenzyme A (HMGCoA) reductase inhibitors(such as a statin), insulin and insulin analogues, GLP-1 and GLP-1analogues (e.g., exendin-4) or amylin. In some embodiments the agent isan immunomodulatory factor such as anti-CD3 mAb, growth factors such asHGF, vascular endothelial growth factor (VEGF), platelet derived growthfactor (PDGF), lactogens, or parathyroid hormone related protein(PTHrP). In one example, the mutated FGF1 protein is administered incombination with a therapeutically effective amount of another FGF, suchas FGF21, FGF19, or both, heparin, or combinations thereof.

In some embodiments, methods are provided for treating diabetes orpre-diabetes in a subject by administering a therapeutically effectiveamount of a composition including an FGFR1c-binding protein multimer ora mutated FGF1 protein (such as a protein generated using the sequencesshown in Tables 1 and 2, the sequences in any of SEQ ID NOS: 21-84,113-120 and 191-238 or those encoding a protein having at least 90%, atleast 95%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100% sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11,12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 173, 174, 175,177, 178, 179, 181, 182, 183, 185, 186, 187, 188, 189, 191, 192, 193,194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207,208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235,236, 237 or 238), or a nucleic acid encoding the FGFR1c-binding proteinmultimer or the mutated FGF1 protein, to the subject. The subject canhave diabetes type I or diabetes type II. The subject can be anymammalian subject, including human subjects and veterinary subjects suchas cats and dogs. The subject can be a child or an adult. The subjectcan also be administered insulin. The method can include measuring bloodglucose levels.

In some examples, the method includes selecting a subject with diabetes,such as type I or type II diabetes, or a subject at risk for diabetes,such as a subject with pre-diabetes. These subjects can be selected fortreatment with the disclosed FGFR1c-binding protein multimer or mutatedFGF1 proteins (such as a protein g generated using the sequences shownin Tables 1 and 2, the sequences in any of SEQ ID NOS: 21-84, 113-120and 191-238 or those encoding a protein having at least 90%, at least95%, at least 95%, at least 96%, at least 97%, at least 98%, at least99% or 100% sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,112, 113, 114, 115, 116, 117, 118, 119, 120, 173, 174, 175, 177, 178,179, 181, 182, 183, 185, 186, 187, 188, 189, 191, 192, 193, 194, 195,196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209,210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223,224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or238) or nucleic acid molecules encoding such.

In some examples, a subject with diabetes may be clinically diagnosed bya fasting plasma glucose (FPG) concentration of greater than or equal to7.0 millimole per liter (mmol/L) (126 milligram per deciliter (mg/dL)),or a plasma glucose concentration of greater than or equal to 11.1mmol/L (200 mg/dL) at about two hours after an oral glucose tolerancetest (OGTT) with a 75 gram (g) load, or in a patient with classicsymptoms of hyperglycemia or hyperglycemic crisis, a random plasmaglucose concentration of greater than or equal to 11.1 mmol/L (200mg/dL), or HbA1c levels of greater than or equal to 6.5%. In otherexamples, a subject with pre-diabetes may be diagnosed by impairedglucose tolerance (IGT). An OGTT two-hour plasma glucose of greater thanor equal to 140 mg/dL and less than 200 mg/dL (7.8-11.0 mM), or afasting plasma glucose (FPG) concentration of greater than or equal to100 mg/dL and less than 125 mg/dL (5.6-6.9 mmol/L), or HbA1c levels ofgreater than or equal to 5.7% and less than 6.4% (5.7-6.4%) isconsidered to be IGT, and indicates that a subject has pre-diabetes.Additional information can be found in Standards of Medical Care inDiabetes—2010 (American Diabetes Association, Diabetes Care 33:S11-61,2010).

In some examples, the subject treated with the disclosed compositionsand methods has HbAlC of greater than 6.5% or greater than 7%.

In some examples, treating diabetes includes one or more of increasingglucose tolerance (such as an increase of at least 5%, at least 10%, atleast 20%, or at least 50%, for example relative to no administration ofthe FGFR1c-binding protein multimer or mutant FGF1), decreasing insulinresistance (for example, decreasing plasma glucose levels, decreasingplasma insulin levels, or a combination thereof, such as decreases of atleast 5%, at least 10%, at least 20%, or at least 50%, for examplerelative to no administration of the FGFR1c-binding protein multimer ormutant FGF1), decreasing serum triglycerides (such as a decrease of atleast 10%, at least 20%, or at least 50%, for example relative to noadministration of the FGFR1c-binding protein multimer or mutant FGF1),decreasing free fatty acid levels (such as a decrease of at least 5%, atleast 10%, at least 20%, or at least 50%, for example relative to noadministration of the FGFR1c-binding protein multimer or mutant FGF1),and decreasing HbA1c levels in the subject (such as a decrease of atleast 0.5%, at least 1%, at least 1.5%, at least 2%, or at least 5% forexample relative to no administration of the FGFR1c-binding proteinmultimer or mutant FGF1). In some embodiments, the disclosed methodsinclude measuring glucose tolerance, insulin resistance, plasma glucoselevels, plasma insulin levels, serum triglycerides, free fatty acids,and/or HbA1c levels in a subject.

In some examples, administration of an FGFR1c-binding protein multimeror a mutated FGF1 protein (such as a protein generated using thesequences shown in Tables 1 and 2, the sequences in any of SEQ ID NOS:21-84, 113-120 and 191-238 or those encoding a protein having at least90%, at least 95%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or 100% sequence identity to SEQ ID NO: 6, 7, 8, 9,10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 173, 174,175, 177, 178, 179, 181, 182, 183, 185, 186, 187, 188, 189, 191, 192,193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220,221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,235, 236, 237 or 238), or nucleic acid molecule encoding such, treats ametabolic disease, such as diabetes (such as type II diabetes) orpre-diabetes, by decreasing of HbAlC, such as a reduction of at least0.5%, at least 1%, or at least 1.5%, such as a decrease of 0.5% to 0.8%,0.5% to 1%, 1 to 1.5% or 0.5% to 2%. In some examples the target forHbAlC is less than about 6.5%, such as about 4-6%, 4-6.4%, or 4-6.2%. Insome examples, such target levels are achieved within about 26 weeks,within about 40 weeks, or within about 52 weeks. Methods of measuringHbA1C are routine, and the disclosure is not limited to particularmethods. Exemplary methods include HPLC, immunoassays, and boronateaffinity chromatography.

In some examples, administration of an FGFR1c-binding protein multimeror a mutated FGF1 protein (such as a protein generated using thesequences shown in Tables 1 and 2, the sequences in any of SEQ ID NOS:21-84, 113-120 and 191-238 or those encoding a protein having at least90%, at least 95%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or 100% sequence identity to SEQ ID NO: 6, 7, 8, 9,10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 173, 174,175, 177, 178, 179, 181, 182, 183, 185, 186, 187, 188, 189, 191, 192,193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220,221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,235, 236, 237 or 238), or nucleic acid molecule encoding such, treatsdiabetes or pre-diabetes by increasing glucose tolerance, for example,by decreasing blood glucose levels (such as two-hour plasma glucose inan OGTT or FPG) in a subject. In some examples, the method includesdecreasing blood glucose by at least 5% (such as at least 10%, at least15%, at least 20%, at least 25%, at least 30%, at least 35%, or more) ascompared with a control (such as no administration of any of insulin, anFGFR1c-binding protein multimer or a mutated FGF1 protein (such as aprotein generated using the sequences shown in Tables 1 and 2, thesequences in any of SEQ ID NOS: 21-84, 113-120 and 191-238 or thoseencoding a protein having at least 90%, at least 95%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or 100% sequenceidentity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 173, 174, 175, 177, 178, 179, 181, 182, 183,185, 186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197, 198, 199,200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227,228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238), or a nucleicacid molecule encoding such). In particular examples, a decrease inblood glucose level is determined relative to the starting blood glucoselevel of the subject (for example, prior to treatment with anFGFR1c-binding protein multimer or a mutated FGF1 protein (such as aprotein generated using the sequences shown in Tables 1 and 2, thesequences in any of SEQ ID NOS: 21-84, 113-120 and 191-238 or thoseencoding a protein having at least 90%, at least 95%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or 100% sequenceidentity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 173, 174, 175, 177, 178, 179, 181, 182, 183,185, 186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197, 198, 199,200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227,228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238), or nucleicacid molecule encoding such). In other examples, decreasing bloodglucose levels of a subject includes reduction of blood glucose from astarting point (for example greater than about 126 mg/dL FPG or greaterthan about 200 mg/dL OGTT two-hour plasma glucose) to a target level(for example, FPG of less than 126 mg/dL or OGTT two-hour plasma glucoseof less than 200 mg/dL). In some examples, a target FPG may be less than100 mg/dL. In other examples, a target OGTT two-hour plasma glucose maybe less than 140 mg/dL. Methods to measure blood glucose levels in asubject (for example, in a blood sample from a subject) are routine.

In other embodiments, the disclosed methods include comparing one ormore indicator of diabetes (such as glucose tolerance, triglyceridelevels, free fatty acid levels, or HbA1c levels) to a control (such asno administration of any of insulin, any FGFR1c-binding protein multimeror any mutated FGF1 protein (such as a protein generated using thesequences shown in Tables 1 and 2, the sequences in any of SEQ ID NOS:21-84, 113-120 and 191-238 or those encoding a protein having at least90%, at least 95%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or 100% sequence identity to SEQ ID NO: 6, 7, 8, 9,10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 173, 174,175, 177, 178, 179, 181, 182, 183, 185, 186, 187, 188, 189, 191, 192,193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220,221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,235, 236, 237 or 238), or a nucleic acid molecule encoding such),wherein an increase or decrease in the particular indicator relative tothe control (as discussed above) indicates effective treatment ofdiabetes. The control can be any suitable control against which tocompare the indicator of diabetes in a subject. In some embodiments, thecontrol is a sample obtained from a healthy subject (such as a subjectwithout diabetes). In some embodiments, the control is a historicalcontrol or standard reference value or range of values (such as apreviously tested control sample, such as a group of subjects withdiabetes, or group of samples from subjects that do not have diabetes).In further examples, the control is a reference value, such as astandard value obtained from a population of normal individuals that isused by those of skill in the art. Similar to a control population, thevalue of the sample from the subject can be compared to the meanreference value or to a range of reference values (such as the high andlow values in the reference group or the 95% confidence interval). Inother examples, the control is the subject (or group of subjects)treated with placebo compared to the same subject (or group of subjects)treated with the therapeutic compound in a cross-over study. In furtherexamples, the control is the subject (or group of subjects) prior totreatment.

The disclosure is illustrated by the following non-limiting Examples.

Example 1 Preparation of Mutated FGF1 Proteins

Mutated FGF1 proteins can be made using known methods (e.g., see Xia etal., PLoS One. 7(11):e48210, 2012). An example is provided below.

Briefly, a nucleic acid sequence encoding an FGF1 mutant protein (e.g.,any of SEQ ID NOs: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 173, 174, 175, 177, 178, 179, 181, 182, 183,185, 186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197, 198, 199,200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227,228, 229, 230, 231, 232, 233, 234, 235, 236, 237 and 238) can be fuseddownstream of an enterokinase (EK) recognition sequence (Asp₄Lys)preceded by a flexible 20 amino acid linker (derived from the S-tagsequence of pBAC-3) and an N-terminal (His)₆ tag. The resultingexpressed fusion protein utilizes the (His)₆ tag for efficientpurification and can be subsequently processed by EK digestion to yieldthe mutant FGF1 protein.

The mutant FGF1 protein can be expressed from an E. coli host afterinduction with isopropyl-β-D-thio-galactoside. The expressed protein canbe purified utilizing sequential column chromatography onNi-nitrilotriacetic acid (NTA) affinity resin followed by ToyoPearlHW-405 size exclusion chromatography. The purified protein can bedigested with EK to remove the N-terminal (His)₆ tag, 20 amino acidlinker, and (Asp4Lys) EK recognition sequence. A subsequent secondNi-NTA chromatographic step can be utilized to remove the releasedN-terminal mutant FGF1 protein (along with any uncleaved fusionprotein). Final purification can be performed using HiLoad Superdex 75size exclusion chromatography equilibrated to 50 mM Na₂PO₄, 100 mM NaCl,10 mM (NH₄)₂SO₄, 0.1 mM ethylenediaminetetraacetic acid (EDTA), 5 mML-Methionine, pH at 6.5 (“PBX” buffer); L-Methionine can be included inPBX buffer to limit oxidization of reactive thiols and other potentialoxidative degradation.

In some examples, the enterokinase is not used, and instead, an FGF1mutant protein (such as one that includes an N-terminal methionine) canbe made and purified using heparin affinity chromatography.

For storage and use, the purified mutant FGF1 protein can be sterilefiltered through a 0.22 micron filter, purged with N₂, snap frozen indry ice and stored at −80° C. prior to use. The purity of the mutant FGF1protein can be assessed by both Coomassie Brilliant Blue and SilverStain Plus (BIO-RAD Laboratories, Inc., Hercules Calif.) stained sodiumdodecylsulfate polyacrylamide gel electrophoresis (SDS PAGE). MutantFGF1 proteins can be prepared in the absence of heparin. Prior to IVbolus, heparin, or PBS, can be added to the protein.

Example 2 N-Terminally Truncated FGF1 Reduces Blood Glucose in Ob/ObMice

We have shown that administration of mature rFGF1 to ob/ob mice canlower blood glucose and have reduced adverse effects as compared tothose observed with thiazolidinediones (TZDs).

To dissociate the mitogenic effects of rFGF1 from its glucose loweringactivities, an FGF1 ligand was generated that lacks the first 24residues from the N-terminus, rFGF1^(ΔNT) (SEQ ID NO: 7). Based on thecrystal structures of FGF1-FGFR complexes, the truncation was predictedto reduce the binding affinity of FGF1 for selected FGFRs includingFGFR4, and hence the ligand's mitogenicity.

Animals

Mice were housed in a temperature-controlled environment with a 12-hourlight/12-hour dark cycle and handled according to institutionalguidelines complying with U.S. legislation. Male ob/ob mice(B6.V-Lep^(ob)/J, Jackson laboratories) and male C57BL/6J mice receiveda standard or high fat diet (MI laboratory rodent diet 5001, HarlanTeklad; high fat (60%) diet F3282, Bio-Serv) and acidified water adlibitum. STZ-induced diabetic mice on the C57BL/6J background werepurchased from Jackson laboratories. 0.1 mg/ml solutions in PBS of mouseFGF1 (Prospec, Ness Ziona, Israel), human FGF1 (Prospec, Ness Ziona,Israel), mouse FGF2 (Prospec, Ness Ziona, Israel), mouse FGF9 (Prospec,Ness Ziona, Israel), and mouse FGF10 (R&D systems) were injected asdescribed.

Serum Analysis

Blood was collected by tail bleeding either in the ad libitum fed stateor following overnight fasting. Free fatty acids (Wako), triglycerides(Thermo) and cholesterol (Thermo) were measured using enzymaticcolorimetric methods following the manufacturer's instructions. Seruminsulin levels were measured using an Ultra Sensitive Insulin ELISA kit(Crystal Chem). Plasma adipokine and cytokine levels were measured usingMilliplex™ MAP and Bio-Plex Pro™ kits (Millipore and Bio-Rad).

Metabolic Studies

Glucose tolerance tests (GTT) were conducted after o/n fasting. Micewere injected i.p. with 1 g of glucose per/kg bodyweight and bloodglucose was monitored at 0, 15, 30, 60, and 120 min using a OneTouchUltra glucometer (Lifescan Inc). Insulin tolerance tests (ITT) wereconducted after 3 h fasting. Mice were injected i.p. with 2 U ofinsulin/kg bodyweight (Humulin R; Eli Lilly) and blood glucose wasmonitored at 0, 15, 30, 60, and 90 min using a OneTouch Ultra glucometer(Lifescan Inc). Real-time metabolic analyses were conducted in aComprehensive Lab Animal Monitoring System (Columbus Instruments). CO₂production, 02 consumption, RQ (relative rates of carbohydrate versusfat oxidation), and ambulatory counts were determined for sixconsecutive days and nights, with at least 24 h for adaptation beforedata recording. Total body composition analysis was performed using anEchoMRI-100™ (Echo Medical Systems, LLC)

Purification of FGF and FGFR Proteins

Human FGF1 (M1 to D155; SEQ ID NO: 2) and N-terminally truncated humanFGF1 (FGF1^(ΔNT) On K25 to D155; SEQ ID NO: 7) were expressed inEscherichia coli cells and purified from the soluble bacterial celllysate fraction by heparin affinity, ion exchange, and size exclusionchromatographies. The minimal ligand-binding domain of human FGFR1c(D142 to R365), FGFR2b (A140 to E369), FGFR2c (N149 to E368), FGFR3c(D147 to E365), and FGFR4 (Q144 to D355) was refolded in vitro frombacterial inclusion bodies and purified by published protocols.

Mice received a standard diet (ob/ob and db/db mice) or high fat diet(C57/BL6 mice, 60% fat, F3282, Bio-Serv) and acidified water ad libitum.Blood glucose levels were monitored either in the ad libitum fed stateor following overnight fasting after injection of recombinant FGF1 orrFGF1^(ΔNT) (in PBS, Prospec, Israel) using the specified delivery routeand dosage. Glucose tolerance tests (GTT) and Insulin tolerance tests(ITT) were conducted after overnight and 5 hour fasting, respectively.Glucose (1 g/kg i.p.) or insulin (0.5 U insulin/kg i.p.) was injectedand blood glucose monitored. Serum analyses were performed on bloodcollected by tail bleeding either in the ad libitum fed state orfollowing overnight fasting.

Remarkably, parenteral delivery of rFGF1^(ΔNT) lowered blood glucoselevels to the same extent as rFGF1 in both genetic- and diet-inducedmouse models of diabetes (FIGS. 2A and 2B). rFGF1^(ΔNT) also retainedthe feeding suppression effects observed with rFGF1 (FIG. 2C).Therefore, the synthetic effects of exogenous rFGF1 on physiology, suchas glucose homeostasis and feeding behavior, differ from and areindependent of its classical role as a growth factor and mitogen.

Example 3 FGF1 Mutant Proteins Reduce Blood Glucose Levels in DiabeticMice

FGF1 mutants shown in Table 3 were tested as described in Example 2.

As shown in Table 3, up to 12 N-terminal amino acids can be deleted fromFGF1 without significantly affecting activity, while an FGF1 mutantlacking 14 N-terminal amino acids failed to lower glucose in diabeticmice. Mutations that increase the thermal stability of FGF1 weregenerally well tolerated, however glucose lowering activity was lost ina mutant with high thermal stability. Furthermore, mutations to theputative heparan sulfate binding site had minimal effect to the glucoselowering actions of FGF1. Notably, the effects of N-terminal deletionsand selected stabilizing mutations appeared additive.

FGF1 (SEQ ID NO:) Relative activity wt FGF1 (5) xxx Reduced mitogenicmutants FGF1^(ΔNT)(10-140αα) (7) xxxx FGF1^(ΔNT2)(14-140αα) (8) inactiveFGF1^(ΔNT3)(12-140αα) (9) xxx (shorter duration) FGF1(1-140αα) K12V,N95V (10) xxxx FGF1(1-140αα) K12V, L46V, E87V, N95V, inactive P134V (11)FGF1(7-140αα) K118N (12) inactive FGF1(7-140αα) K118E (13) inactiveStabilizing Mutants FGF1(1-140αα)K12V, P134V, C117V (22) xxxFGF1(1-140αα) L44F, C83T, C117V, xxx F132W (28) FGF1(1-140αα) L44F,M67I, L73V, V109L, xxx (shorter duration) L111I, C117V, A103G, R119G,Δ104-106, Δ120-122 (40) FGF1(1-140αα) K12V, N95V, C117V (54) xxx (longerduration) FGF1(1-140αα) K12V, L46V, E87V, N95V, inactive P134V, C117V(212) Heparan binding site mutations FGF1^(ΔHBS) K112D, K113Q, K118V(113) xx FGF1^(ΔHBS) (1-140αα) K112D, K113Q, xx K118V (226) Combinationmutations FGF^(ΔNT1C) (10-140αα) K12V, N95V, xxxx (longer duration)C117V (225) FGF1 (1-140αα) K12V, Q40P, S47I, H93G, xx N95V (227)FGF1^(ΔNT1)(10-140αα) K12V, Q40P, S47I, H93G, xx N95V (228) FGF1(1-140αα) K12V, L44F, C83T, N95V, xxx C117V, F132W (229) ChimeraswtFGF1^(ΔHBS)-FGF21^(C-tail) (219) xx wtFGF1^(ΔHBS)-FGF19^(C-tail) (220)xx

Example 4 Effect on Intracellular Signaling with FGF1 Mutants

Peptides M1, M2, M3, M4, and M5 (see SEQ ID NO: 22, 28, 40, 54 and 212,respectively); KN (SEQ ID NO: 10), KLE (SEQ ID NO: 11), and FGF1 (SEQ IDNO: 5) were generated as described in Example 1. The NT truncations,peptides NT1 (SEQ ID NO: 7), NT2 (SEQ ID NO: 8), and NT3 (SEQ ID NO: 9),were prepared without the His tag and enterokinase cleavage, andpurified with heparin affinity and ion exchange chromatography. Peptides(10 ng/ml) were incubated with serum-starved HEK293 cells for 15minutes. Total cell lysates were subject to Western blotting withantibodies specific for pAkt, Akt, pERK and ERK.

As shown in FIG. 8, the thermostable M3 analog shows reduced ERKsignaling, similar to that seen with the M5 analog, correlating with thereduced glucose lowering activity seen in ob/ob mice.

As shown in FIG. 9, deletion of 9 (NT1) or 11 (NT3) N-terminal residuesof FGF1 does not significantly affecting FGFR downstream signaling,while deletion of 13 (NT2) residues severely compromises ERKphosphorylation. The introduction of the point mutations K12V, N95Vreduced ERK phosphorylation, while incorporating the additionalmutations L46V, E87V and P134V totally abrogates ERK signaling.

As shown in FIG. 10, deletion of 9 amino acids from the N-terminus ofFGF1 (NT1, FGF1^(ΔNT)) induces an ˜100 fold reduction in FGFR signaling,as seen in the reduced phosphorylation of downstream ERK and AKTpathways.

Example 5 Effect on Blood Glucose with FGF1 Mutants

Peptides M1, M2, M3 (see SEQ ID NOS: 22, 28, and 40, respectively), FGF1(SEQ ID NO: 5), NT1 (SEQ ID NO: 7) and NT2 (SEQ ID NO: 8) were generatedas described in Example 4. Peptides (0.5 mg/kg) or PBS (control) wereinjected SQ into 5 mo old C57BL/6J ob/ob mice fed normal chow. Bloodglucose levels were subsequently determined.

As shown in FIGS. 11A and 11B, peptides M1 and M2 lowered glucose aswell as wild-type FGF1. Thus, FGF1 analogs can be designed withincreased thermostability, and improved pharmacokinetic properties,while still having desired effects on lowing blood glucose. Thus, theFGF1 portion of the FGF2/FGF1 chimeras provided herein can include thesemutations (e.g., one or more of K12V, C117V, P134V, L44F, C83T, andF132W).

As shown in FIG. 12 peptide FGF1^(ΔNT) (NT1) significantly loweredglucose, while FGF1^(ΔNT2) (NT2) lost its ability to significantlylowered glucose. Thus, FGF1 can be N-terminally truncated (such as thefirst 9 amino acids, but not more than 13 amino acids), while stillhaving desired effects on lowing blood glucose. Thus, the FGF1 portionof the FGF2/FGF1 chimeras provided herein can include such a truncation.

Example 6 Glucose Lowering Correlates with FGFR Signaling

Peptides FGF1 (SEQ ID NO: 5), FGF1^(ΔNT) (SEQ ID NO: 7) and NT2 (SEQ IDNO: 8) were generated as described in Example 4. Peptides (10 ng/ml)were incubated with serum-starved HEK293 cells for 15 minutes. Totalcell lysates were subject to Western blotting with antibodies specificfor pAkt, Akt, pERK and ERK.

As shown in FIG. 13, comparable activation of the downstream signalingeffectors ERK and AKT is seen with FGF1 and two independent preparationsof FGF1^(ΔNT) that lacks the N-terminal 9 amino acids (SEQ ID NO: 7). Incontrast, the deletion of an additional 4 N-terminal amino acidsmarkedly reduces both ERK and AKT phosphorylation. These in vitroFGFR-mediated signaling results correlate with the in vivo glucoselowering effect observed in FIG. 12, supporting the hypothesis that theglucose-lowering activity is mediated through an FGF receptor.

Example 7 Effect on Blood Glucose with FGF1 Mutants

Peptides FGF1-KLE (SEQ ID NO: 11) or FGF1-KN (SEQ ID NO: 10) weregenerated as described in Example 1. Peptides (0.5 mg/kg) were injectedSQ into 5 mo old C57BL/6J ob/ob mice fed normal chow. Blood glucoselevels were subsequently determined 0 to 120 hours later.

As shown in FIG. 14, the FGF1-KN mutant retained the ability to lowerglucose for 120 hrs despite a marked reduction in its mitogenicactivity. In contrast, the mitogenically dead FGF1-KLE failed to lowerglucose. These results indicate that the mitogenicity andglucose-lowering activity can be independently affected through targetedmutations. Thus, the FGF1 portion of the FGF2/FGF1 chimeras providedherein can include the mutations in the KN mutant (e.g., one or more ofK12V and N95V) to reduce its mitogenicity without significantlycompromising its ability to lower blood glucose levels.

Example 8 Dose-Response Effects on Blood Glucose with FGF1 Mutants

Peptides rFGF1^(ΔNT) (SEQ ID NO: 7) and rFGF1 (SEQ ID NO: 5) weregenerated as described in Example 4 (generated with an N-terminalmethionine and purified with heparin affinity and ion exchangechromatography). Peptides (0.016 to 10 ng/ml) or PBS were incubated withserum-starved HEK293 cells for 15 minutes. Total cell lysates weresubject to Western blotting with antibodies specific for pFRS2α, pAkt,Akt, pERK and ERK. Peptides (0.5 mg/kg) were injected SQ into high fatdiet (HFD) fed diet-induced obesity (DIO) mice or into 12 week oldC57BL/6J ob/ob mice (0 to 0.5 mg/kg) fed normal chow. Blood glucoselevels were subsequently determined.

As shown in FIG. 15A, deletion of 9 N-terminal amino acids of FGF1significantly reduces FGFR downstream signaling, includingphosphorylation of ERK and AKT. Dose dependent phosphorylation of theFGFR substrate FRS2a, confirms that both FGF1 and FGF1^(ΔNT) are capableof activating FGF receptors.

As shown in FIG. 15B, food intake in DIO mice after receiving, rFGF1 orrFGF1^(ΔNT) was significantly reduced, as compared to mice that receivedPBS alone. The similarity in the extent of the transient reduction infood intake between rFGF1 and rFGF1^(ΔNT) further supports theconclusion that both proteins achieve their in vivo glucose loweringeffects by signaling through an FGF receptor.

As shown in FIG. 15C, an essentially identical dose-response curve wasobserved for the glucose lowering effects of rFGF1 and rFGF1^(ΔNT) (NT1)in ob/ob mice. Given the significant reduction in mitogenicity ofrFGF1^(ΔNT), these results demonstrate that the glucose lowering andmitogenic activities of FGF1 can be dissociated.

Example 9 Effect of N-terminal FGF1 Truncations on Blood Glucose Levels

Peptides NT1 (SEQ ID NO: 7), NT2 (SEQ ID NO: 8), and NT3 (SEQ ID NO: 9)were generated as described in Example 4. Peptides (0.5 mg/kg) wereinjected SQ into 5 mo old C57BL/6J ob/ob mice fed normal chow, orpeptides (0 to 0.5 mg/kg) were injected SQ into 12 week old ob/ob micefed normal chow. Blood glucose levels were subsequently determined (0hr, 16 hrs, or 24 hrs).

As shown in FIG. 16 if the N-terminus is truncated at 14 amino acids,glucose lowering ability is dramatically decreased (NT2). Thus, FGF1 canbe N-terminally truncated (such as the first 9, 10, or 11 amino acids),while maintaining the desired effects on lowering blood glucose. Thus,the FGF1 mutants provided herein can include such a truncation.

In another experiment, NT1 (SEQ ID NO: 7) (0.5 mg/kg) was injected SQinto 8 month old HFD-fed wildtype (FGFR1 f/f, open bars) oradipose-specific FGFR1 knockout (R1KO, aP2-Cre; FGFR1 f/f, filled bars)mice Blood glucose levels were subsequently determined (0 hr, 12 hrs, or24 hrs).

As shown in FIGS. 17A and 17B, rFGF1^(ΔNT) (NT1) (SEQ ID NO: 7) lowersblood glucose levels in HFD-fed wildtype mice (control) but has noeffect on FGFR1 KO (mutant) mice. FIG. 17A reports the changes in bloodglucose, while FIG. 17B reports the data normalized to starting glucoselevels at 100%. These results demonstrate that expression of FGFR1 inadipose tissue is required for rFGF1^(ΔNT) mediated glucose lowering.

As shown in FIGS. 18A and 18B mouse rFGF1 (amino acids 1-15 of SEQ IDNO: 4) lowers blood glucose levels in HFD-fed wildtype mice (FGFR1 f/fmice, filled bars) but has no effect on aP2-Cre; FGFR1 f/f (FGFR1KO,speckled bars) mice. FIG. 17A reports the changes in blood glucose,while FIG. 17B reports the data normalized to starting glucose levels at100%. These results demonstrate that expression of FGFR1 in adiposetissue is required for rFGF1 mediated glucose lowering.

Example 10 Effect of FGF1 Point Mutations on Blood Glucose Lowering

Peptides K118E (SEQ ID NO: 13), K118N (SEQ ID NO: 12), FGF1 (SEQ ID NO:5), and KKK (SEQ ID NO: 114) were generated as described in Example 1,while FGF1^(ΔNT) (NT1) (SEQ ID NO: 7) was expressed with an N-terminalmethionine and purified using heparin affinity and ion exchangechromatography. Peptides (0.5 mg/kg) or PBS were injected SQ into 7months HFD-fed C57BL/6J mice. Blood glucose levels were subsequentlydetermined (0 to 120 hours). These mice are diet-induced obese (DIO)mice.

As shown in FIG. 19, mutation of the single lysine, K118, to either Asn(K118N (SEQ ID NO: 12)) or Glu (K118E (SEQ ID NO: 13)), is sufficient toabrogate glucose lowering activity in DIO mice.

As shown in FIGS. 21 and 22, mutating selected amino acids implicated inthe heparin binding site of FGF1, namely amino acids K112, K113, andK118, resulted in a mutated FGF1 sequence that could lower blood glucoselevels in ob/ob mice. Thus, the FGF1 mutants provided herein can includemutations at all three of K112, K113, and K118, such as a K112D, K113Q,and K118V substitution. However, while the mutation of K118 to thehydrophobic residue valine was tolerated, mutations involving a chargereversal (K118E) or to a polar residue (K118N) are not tolerated.

Example 11 Effect of FGF1 Point Mutations on Blood Glucose Lowering

Peptides KN (SEQ ID NO: 10), KKK (Salk_(—)010, SEQ ID NO: 226), FGF1(SEQ ID NO: 5), and KLE (Salk_(—)011, SEQ ID NO: 11) were generated asdescribed in Example 1, while FGF1^(ΔNT) (NT1) (SEQ ID NO: 7) andFGF1^(ΔNTKN) (Salk_(—)009, SEQ ID NO: 225) were expressed with anN-terminal methionine and purified using heparin affinity and ionexchange chromatographies. Peptides (0.5 mg/kg) or PBS were injected SQinto 7 months HFD-fed C57BL/6J mice. Blood glucose levels weresubsequently determined (0 to 120 hours).

As shown in FIGS. 32A and 32B, combining the mutations K12V and N95Vwith the deletion of the N-terminal residues resulted in a mutated FGF1sequence (SEQ ID NO: 225) that could lower blood glucose levels in ob/obmice. The combined mutations of K12V N95V with the stabilizing mutationsQ40P S471 H93G (SEQ ID NO: 11) also lowered blood glucose levels inob/ob mice. Transient reductions in food intake were observed withselected FGF1 analogs (FIGS. 32C and 32D). Furthermore, a singleinjection of SEQ ID NO: 225 was able to sustain the low glucose levelsfor more than 7 days.

Example 12 Effect of FGF1 Point Mutations on Blood Glucose Lowering

Peptides FGF1 (SEQ ID NO: 5), and Salk_(—)013 (SEQ ID NO: 31) weregenerated as described in Example 1, while Salk_(—)012 (SEQ ID NO: 79)was expressed with an N-terminal methionine and purified using heparinaffinity and ion exchange chromatographies. Peptides (0.5 mg/kg) or PBSwere injected SQ into 7 months HFD-fed C57BL/6J mice. Blood glucoselevels were subsequently determined (0 to 96 hours).

As shown in FIG. 33A, combining the mutations K12V N95V with thestabilizing mutations Q40P S471 H93G and the deletion of the N-terminalresidues resulted in a mutated FGF1 sequence (Salk_(—)012) that couldlower blood glucose levels in ob/ob mice. The combined mutations of K12VN95V with the stabilizing mutations L44F, C83T, C117V, and F132W(Salk_(—)013) also lowered blood glucose levels in ob/ob mice.Salk_(—)012 and Salk_(—)013 has minimal effects on food intake (FIG.33B).

Example 13 Effect of FGF1 Point Mutations on Blood Glucose Lowering

Peptides Salk_(—)014 (SEQ ID NO: 230), Salk_(—)024 (SEQ ID NO: 84),Salk_(—)025 (SEQ ID NO: 208), and Salk_(—)026 (SEQ ID NO: 209), weregenerated as described in Example 1, while Salk_(—)023 (SEQ ID NO: 38)was expressed with an N-terminal methionine and purified using heparinaffinity and ion exchange chromatographies. Peptides (0.5 mg/kg) or PBSwere injected SQ into 7 months HFD-fed C57BL/6J mice. Blood glucoselevels were subsequently determined (0 to 72 hours).

As shown in FIG. 34A, FGF1 with the stabilizing point mutation C117Valone, or in combination with additional mutations reduces blood glucosein ob/ob mice for more than 72 hours. Various transient effects on foodintake in the 24 hours after injection were observed (FIG. 34B).

Example 14 Effect of FGF1 Point Mutations on Blood Glucose Lowering

Peptides Salk_(—)014 (SEQ ID NO: 230), Salk_(—)022 (SEQ ID NO: 119), andSalk_(—)027 (SEQ ID NO: 207) were generated as described in Example 1.Peptides (0.5 mg/kg) or PBS were injected SQ into 7 months HFD-fedC57BL/6J mice. Blood glucose levels were subsequently determined (0 to24 hours).

As shown in FIG. 35A, FGF1 with the stabilizing point mutation C117Valone, or in combination with additional mutations that affect theability to bind to heparan sulfate proteoglycans (HSPG), reduce bloodglucose in ob/ob mice. The transient suppression of food intake is lostin analogs incorporating mutations that affect HSPG binding (FIG. 35B).

Example 15 Effect of FGF1 Point Mutations on Blood Glucose Lowering

Peptides Salk_(—)014 (SEQ ID NO: 230), was generated as described inExample 1, while Salk_(—)032 (SEQ ID NO: 215) was expressed with anN-terminal methionine and purified using heparin affinity and ionexchange chromatographies. Peptides (0.5 mg/kg) or PBS were injected SQinto 7 months HFD-fed C57BL/6J mice. Blood glucose levels weresubsequently determined (0 to 24 hours).

As shown in FIG. 36A, the K12V N95V mutations, the stabilizing cysteinemutations C12T, C83S C117V, and the deletion of the N-terminal residuescan be combined to generate an FGF1 analog that is able to robustlyreduce blood glucose levels with only minor effects on feeding (FIG.36B), indicating that these effects can be differentiated.

Example 16 Effect of FGF1-FGF19 Chimeric Proteins on Blood GlucoseLowering

Peptides Salk_(—)014 (SEQ ID NO: 230), and Salk_(—)019 (SEQ ID NO: 224)were generated as described in Example 1. Peptides (Salk_(—)014; 0.5mg/kg, Salk_(—)019; indicated doses) or PBS were injected SQ into 7months HFD-fed C57BL/6J mice. Blood glucose levels were subsequentlydetermined (0 to 24 hours).

As shown in FIGS. 37A and 37B, the chimeric protein (SEQ ID NO: 224)generated by fusing the β-klotho binding domain of FGF19 (SEQ ID NO:100) to the C-terminus of FGF1 failed to affect blood glucose or tosuppress food intake, even at a 5 fold higher concentration (2.5 mg/kgcompared to 0.5 mg/kg). Given that a chimeric protein composed of FGF1and the β-klotho binding region of FGF21 was active in lowering bloodglucose, these results indicate that this analog is being targeted toFGFR4-β-klotho receptor complex that does not affect blood glucoselevels.

Example 17 Effect of FGF1-FGF21 chimeric Proteins on Blood GlucoseLowering

Peptides FGF1 (SEQ ID NO: 5), FGF1^(ΔNT) (SEQ ID NO: 7), FGF21 (SEQ ID20) and FGF1-FGF21 chimera (wherein the FGF1 portion includes K112D,K113Q, and K118V mutations, thus the chimera is SEQ ID NO: 114+SEQ IDNO: 86) were generated as described in Example 1. Peptides (0.5 mg/kg,)or PBS were injected SQ into 7 months HFD-fed C57BL/6J mice. Bloodglucose levels were subsequently determined (0 to 48 hours).

As shown in FIG. 38, wildtype FGF21 weakly lowers glucose compared towildtype FGF1, and FGF1^(ΔNT). In contrast, a chimeric proteinconstructed from a mutant FGF1 fused to the β-klotho binding region ofFGF21 (FGF1-FGF21^(C-tail)) was effective at lowering blood glucose.

In view of the many possible embodiments to which the principles of thedisclosure may be applied, it should be recognized that the illustratedembodiments are only examples of the disclosure and should not be takenas limiting the scope of the invention. Rather, the scope of thedisclosure is defined by the following claims. We therefore claim as ourinvention all that comes within the scope and spirit of these claims.

1. A method of reducing blood glucose in a mammal, comprising: (a) administering a therapeutically effective amount of a mutated mature fibroblast growth factor (FGF) 1 protein to the mammal, or a nucleic acid molecule encoding the mutated FGF1 protein or a vector comprising the nucleic acid molecule, thereby reducing the blood glucose, wherein the mutated mature FGF1 protein comprises: a deletion of at least six contiguous N-terminal amino acids; at least one point mutation; or combinations thereof; (b) administering a therapeutically effective amount of a fibroblast growth factor receptor (FGFR) 1c-binding protein to the mammal, or a nucleic acid molecule encoding the FGFR1c-binding protein or a vector comprising the nucleic acid molecule, thereby reducing the blood glucose, wherein the FGFR1c-binding protein comprises a multimer of FGFR1c-binding proteins; or (c) combinations of (a) and (b).
 2. A method of treating a metabolic disease in a mammal, comprising: (a) administering a therapeutically effective amount of a mutated mature fibroblast growth factor (FGF) 1 protein to the mammal, or a nucleic acid molecule encoding the mutated FGF1 protein or a vector comprising the nucleic acid molecule, thereby treating the metabolic disease, wherein the mutated mature FGF1 protein comprises: a deletion of at least six contiguous N-terminal amino acids; at least one point mutation; or combinations thereof; (b) administering a therapeutically effective amount of a fibroblast growth factor receptor (FGFR) 1c-binding protein to the mammal, or a nucleic acid molecule encoding the FGFR1c-binding protein or a vector comprising the nucleic acid molecule, thereby treating the metabolic disease, wherein the FGFR1c-binding protein comprises a multimer of FGFR1c-binding proteins; or (c) combinations of (a) and (b).
 3. The method of claim 2, wherein the metabolic disease is type 2 diabetes, non-type 2 diabetes, type 1 diabetes, polycystic ovary syndrome (PCOS), metabolic syndrome (MetS), obesity, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), hyperlipidemia, hypertension, latent autoimmune diabetes (LAD), or maturity onset diabetes of the young (MODY).
 4. A method of reducing fed and fasting blood glucose, improving insulin sensitivity and glucose tolerance, reducing systemic chronic inflammation, ameliorating hepatic steatosis in a mammal, reducing food intake, or combinations thereof, comprising: (a) administering a therapeutically effective amount of a mutated mature FGF1 protein to the mammal, or a nucleic acid molecule encoding the mutated FGF1 protein or a vector comprising the nucleic acid molecule, thereby reducing fed and fasting blood glucose, improving insulin sensitivity and glucose tolerance, reducing systemic chronic inflammation, ameliorating hepatic steatosis in a mammal, reducing food intake, or combinations thereof, wherein the mutated mature FGF1 protein comprises: a deletion of at least six contiguous N-terminal amino acids; at least one point mutation; or combinations thereof; (b) administering a therapeutically effective amount of a fibroblast growth factor receptor (FGFR) 1c-binding protein to the mammal, or a nucleic acid molecule encoding the FGFR1c-binding protein or a vector comprising the nucleic acid molecule, thereby reducing fed and fasting blood glucose, improving insulin sensitivity and glucose tolerance, reducing systemic chronic inflammation, and ameliorating hepatic steatosis in a mammal, or combinations thereof, wherein the FGFR1c-binding protein comprises a multimer of FGFR1c-binding proteins; or (c) combinations of (a) and (b).
 5. The method of claim 1, wherein the therapeutically effective amount of the protein is at least 0.5 mg/kg.
 6. The method of claim 1, wherein the administering is subcutaneous, intraperitoneal, intramuscular, or intravenous.
 7. The method of claim 1, wherein the mammal is a cat or dog.
 8. The method of claim 1, wherein the mammal is a human.
 9. The method of claim 1, wherein the mutated mature FGF1 protein comprises a deletion of at least 9, at least 10, at least 11, at least 12 or at least 13 contiguous N-terminal amino acids, wherein the mutated FGF1 protein has reduced mitogenic activity as compared to wild-type mature FGF1 protein.
 10. The method of claim 1, wherein the at least one point mutation comprises a mutation at one or more of K9, K10, K12, L14, Y15, C16, H21, R35, Q40, L44, L46, S47, E49, Y55, M67, L73, C83, L86, E87, H93, Y94, N95, H102, A103, E104, K105, N106, F108, V109, L111, K112, K113, C117, K118, R119, G120, P121, R122, F132, L133, P134, L135, wherein the numbering refers to the sequence shown SEQ ID NO: 5, and wherein the mutated FGF1 protein has reduced mitogenic activity as compared to wild-type mature FGF1 protein.
 11. The method of claim 1, wherein the at least one point mutation comprises one or more of the mutations shown in Table 1, wherein the mutated FGF1 protein has reduced mitogenic activity as compared to wild-type mature FGF1 protein.
 12. The method of claim 1, wherein the at least one point mutation comprises: mutations at K112, K113, and K118, wherein the numbering refers to SEQ ID NO: 5; or replacing amino acid sequence ILFLPLPV (amino acids 145-152 of SEQ ID NO: 2 and 4) to AAALPLPV (SEQ ID NO: 14), ILALPLPV (SEQ ID NO: 15), ILFAPLPV (SEQ ID NO: 16), or ILFLPAPA (SEQ ID NO: 17), wherein the mutated FGF1 protein has reduced mitogenic activity as compared to wild-type mature FGF1 protein.
 13. The method of claim 1, wherein deletion of at least six contiguous N-terminal amino acids further comprises replacing at least 1 of the deleted N-terminal amino acids with a corresponding amino acid from FGF21.
 14. The method of claim 1, wherein a wild-type mature FGF1 protein comprises SEQ ID NO:
 5. 15. The method of claim 1, wherein the mutated mature FGF1 protein comprises at least 95% sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 113, 114, 115, 116, 117, 118, 119, 120, 173-175, 177-179, 181-183, 185-189, and 191-238.
 16. The method of claim 1, wherein the mutated mature FGF1 protein is part of a chimeric protein further comprising (i) a C-terminal region of FGF21 (ii) a C-terminal region of FGF19, (iii) a β-Klotho-binding protein, (iv) a FGFR1c-binding protein, or (v) a FGFR1c-binding protein and a β-Klotho-binding protein.
 17. The method of claim 16, wherein the chimeric protein comprises SEQ ID NO: 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 173, 174, 175, 177, 178, 179, 181, 182, 183, 185, 186, 187, 188, or
 189. 18. The method of claim 1, wherein the FGFR1c-binding protein comprises multimers, wherein at least one monomer of the multimer comprises at least 90% sequence identity SEQ ID NO: 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, or
 190. 19. The method of claim 1, wherein the method further comprises administering an additional therapeutic compound.
 20. The method of claim 19, wherein the additional therapeutic compound is an alpha-glucosidase inhibitor, amylin agonist, dipeptidyl-peptidase 4 (DPP-4) inhibitor, meglitinide, sulfonylurea, or a peroxisome proliferator-activated receptor (PPAR)-gamma agonist.
 21. The method of claim 20, wherein the PPAR-gamma agonist is a thiazolidinedione (TZD), aleglitazar, farglitazar, muraglitazar, or tesaglitazar.
 22. The method of claim 21, wherein the TZD is pioglitazone, rosiglitazone, rivoglitazone, or troglitazone.
 23. An isolated mutated mature fibroblast growth factor (FGF) 1 protein comprising at least 90% sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 173, 174, 175, 177, 178, 179, 181, 182, 183, 185, 186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or
 238. 24. The isolated protein of claim 23, wherein the N-terminal amino acid is a methionine.
 25. The isolated protein of claim 23, wherein the protein is modified to decrease binding affinity for heparin and/or heparan sulfate compared to an FGF1 protein without the modification.
 26. The isolated protein of claim 23, wherein the protein comprises a chimeric protein further comprising (i) a C-terminal region of FGF21 (ii) a C-terminal region of FGF19, (iii) a β-Klotho binding protein, (iv) a FGFR1c-binding protein, or (v) a FGFR1c-binding protein and a β-Klotho-binding protein.
 27. The isolated protein of claim 26, wherein (i) the C-terminal region of FGF21 consists of SEQ ID NO: 86, (ii) the C-terminal region of FGF19 consists of SEQ ID NO: 100, (iii) the β-Klotho binding protein comprises SEQ ID NO: 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145 or 146, or (iv) the FGFR1c-binding protein comprises SEQ ID NO: 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167 or
 190. 28. An isolated nucleic acid encoding the isolated protein of claim
 23. 29. A nucleic acid vector comprising the isolated nucleic acid of claim
 28. 30. A host cell comprising the nucleic acid vector of claim
 29. 31. The isolated protein of claim 23, wherein the protein comprises a mutation at K12 and/or C117, wherein the amino acid numbering is based on SEQ ID NO:
 5. 32. The isolated protein of claim 31, wherein the protein comprises a K12V and/or a C117V mutation, wherein the amino acid numbering is based on SEQ ID NO:
 5. 33. The isolated protein of claim 31, wherein the protein further comprises a mutation at N95 and/or C83, wherein the amino acid numbering is based on SEQ ID NO:
 5. 34. The isolated protein of claim 33, wherein the protein comprises an N95V and/or a C83T mutation, wherein the amino acid numbering is based on SEQ ID NO:
 5. 